U.S. patent number 7,846,412 [Application Number 10/988,923] was granted by the patent office on 2010-12-07 for bioconjugated nanostructures, methods of fabrication thereof, and methods of use thereof.
This patent grant is currently assigned to Emory University. Invention is credited to Xiaohu Gao, Shuming Nie.
United States Patent |
7,846,412 |
Nie , et al. |
December 7, 2010 |
Bioconjugated nanostructures, methods of fabrication thereof, and
methods of use thereof
Abstract
Nanostructures, methods of preparing nanostructures, methods of
detecting targets in subjects, and methods of treating diseases in
subjects, are disclosed. An embodiment, among others, of the
nanostructure includes a quantum dot and a hydrophobic protection
structure. The hydrophobic protection structure includes a capping
ligand and an amphiphilic copolymer, where the hydrophobic
protection structure encapsulates the quantum dot.
Inventors: |
Nie; Shuming (Atlanta, GA),
Gao; Xiaohu (Decatur, GA) |
Assignee: |
Emory University (Atlanta,
GA)
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Family
ID: |
34681665 |
Appl.
No.: |
10/988,923 |
Filed: |
November 15, 2004 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20050136258 A1 |
Jun 23, 2005 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60532028 |
Dec 22, 2003 |
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Current U.S.
Class: |
423/414; 702/20;
438/52; 439/87; 438/466; 702/19; 438/780 |
Current CPC
Class: |
G01N
33/588 (20130101); B82Y 5/00 (20130101); H01F
1/0054 (20130101); A61P 35/00 (20180101); A61K
49/0017 (20130101); A61K 47/6923 (20170801); B82Y
15/00 (20130101); B82Y 25/00 (20130101); A61K
49/0067 (20130101); Y10T 428/2982 (20150115); Y02A
90/10 (20180101); Y02A 90/26 (20180101) |
Current International
Class: |
C01B
31/00 (20060101); H01L 21/00 (20060101); H01L
21/326 (20060101); H01L 21/469 (20060101); G01N
33/48 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
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Primary Examiner: Zhou; Shubo (Joe)
Attorney, Agent or Firm: Thomas, Kayden, Horstemeyer &
Risley, LLP
Government Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under Grant No.:
R01 GM 60562 awarded by the National Institute of Health. The
government has certain rights in the invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/532,028, entitled "BIOCONJUGATED
NANOSTRUCTURES, METHODS OF FABRICATION THEREOF, AND METHODS OF USE
THEREOF" filed on Dec. 22, 2003, the entirety of which is hereby
incorporated by reference.
Claims
Therefore, having thus described the invention, at least the
following is claimed:
1. A nanostructure, comprising: a quantum dot; and a hydrophobic
protection structure encapsulating the quantum dot, said
hydrophobic protection structure comprising: a capping ligand
disposed on the surface of the quantum dot, and an amphiphilic ABC
triblock copolymer, wherein the amphiphilic ABC triblock copolymer
includes a plurality of alkyl side chains conjugated thereto, said
alkyl side-chains each comprising at least eight carbons (--C-8-)
and interacting with the capping ligand, and said amphiphilic ABC
triblock copolymer encapsulating the quantum dot having the capping
ligand disposed thereon and not extending from the surface of said
quantum dot having the capping ligand disposed thereon surface,
thereby forming a hydrophobic protection layer encapsulating the
quantum dot.
2. The nanostructure of claim 1, wherein the amphiphilic ABC
triblock copolymer comprises a poly-butylacrylate segment, a
poly-ethylacrylate segment, and a poly-methacrylic acid segment,
and the plurality of alkyl side-chains are conjugated to the
poly-methacrylic acid segment.
3. The nanostructure of claim 1, wherein the amphiphilic ABC
triblock copolymer has a molecular weight of about 10.sup.5 daltons
and comprises about 77 weight percent of the poly-butylacrylate
segment and about 23 weight percent of the poly-ethylacrylate
segment and the poly-methacrylic acid segment, and wherein the
plurality of alkyl side-chains are conjugated to the
poly-methacrylic acid segment.
4. The nanostructure of claim 1, wherein the quantum dot is
CdTe/CdSe.
5. The nanostructure of claim 1, further comprising a
bio-compatibility compound substantially disposed on the surface of
the amphiphilic ABC triblock copolymer.
6. The nanostructure of claim 5, wherein the bio-compatibility
compound is a polyethylene glycol.
7. The nanostructure of claim 5, further comprising a probe
substantially disposed on the surface of the hydrophobic protection
structure, wherein the probe is an antibody, a polypeptide, a
polynucleotide, a drug molecule, an inhibitor compound, or any
combination thereof.
8. The nanostructure of claim 5, wherein the probe includes a
tumor-targeting ligand.
9. The nanostructure of claim 5, wherein the probe includes a
prostate tumor-targeting ligand.
10. The nanostructure of claim 1, wherein the capping ligand
comprises tri-octylphosphine oxide.
11. A method of preparing a nanostructure, comprising: (i)
providing a quantum dot having a capping ligand disposed on the
surface thereof; and (ii) contacting the quantum dot having a
capping ligand disposed on the surface thereof with a composition
comprising an amphiphilic ABC triblock copolymer, wherein the
amphiphilic ABC triblock copolymer includes a plurality of alkyl
side chains conjugated thereto, said alkyl side-chains each
comprising at least eight carbons (--C-8-), and said amphiphilic
ABC triblock copolymer encapsulating the quantum dot having a
capping ligand disposed on the surface thereof and not extending
from the surface of said quantum dot having a capping ligand
disposed on the surface thereof surface, thereby forming a
hydrophobic protection layer encapsulating the quantum dot.
12. The method of claim 11, further comprising attaching a
bio-compatibility compound to the hydrophobic protection layer.
13. The method of claim 12, wherein the bio-compatibility compound
is a polyethylene glycol.
14. The method of claim 11, further comprising attaching a probe to
the hydrophobic protection layer.
15. The method of claim 14, wherein the probe is an antibody, a
polypeptide, a polynucleotide, a drug molecule, an inhibitor
compound, or any combination thereof.
16. The method of claim 11, wherein the capping ligand comprising
tri-octylphosphine oxide, and wherein the amphiphilic ABC triblock
copolymer structure comprises a poly-butylacrylate segment, a
poly-ethylacrylate segment, and a poly-methacrylic acid
segment.
17. The method of claim 16, wherein the amphiphilic ABC triblock
copolymer has a molecular weight of about 10.sup.5 daltons and
comprises about 77 weight percent of the poly-butylacrylate segment
and about 23 weight percent of the poly-ethylacrylate segment and
the poly-methacrylic acid segment, and wherein the plurality of
alkyl side-chains are conjugated to the poly-methacrylic acid
segment.
Description
FIELD OF THE INVENTION(S)
The present disclosure relates generally to nanostructures, and
relates more particularly, to bioconjugated nanostructures.
BACKGROUND
Recent advances have shown that nanometer-sized semiconductor
particles can be covalently linked with biorecognition molecules
such as peptides, antibodies, nucleic acids, or small-molecule
ligands for applications as fluorescent probes. In comparison with
organic fluorophores, these quantum-confined particles or quantum
dots (QDs) exhibit unique optical and electronic properties, such
as size- and composition-tunable fluorescence emission from visible
to infrared wavelengths, large absorption coefficients across a
wide spectral range, and very high levels of brightness and
photostability. Due to their broad excitation profiles and
narrow/symmetric emission spectra, high-quality QDs are also well
suited for optical multiplexing, in which multiple colors and
intensities are combined to encode genes, proteins, and
small-molecule libraries.
Therefore, the development of high-sensitivity and high-specificity
probes beyond the intrinsic limitations of organic dyes and
fluorescent proteins is of considerable interest to many areas of
research, ranging from molecular and cellular biology to molecular
imaging and medical diagnostics.
SUMMARY
Briefly described, embodiments of this disclosure, among others,
include nanostructures, methods of preparing nanostructures,
methods of detecting targets in subjects, and methods of treating
diseases in subjects. An embodiment, among others, of the
nanostructure includes a quantum dot and a hydrophobic protection
structure. The hydrophobic protection structure includes a capping
ligand and an amphiphilic copolymer, where the hydrophobic
protection structure encapsulates the quantum dot.
Another embodiment of the nanostructure includes at least one
nanospecies and a hydrophobic protection structure. The hydrophobic
protection structure includes at least one compound selected from a
capping ligand, an amphiphilic copolymer, and combinations thereof,
where the hydrophobic protection structure encapsulates the
nanospecies.
An embodiment, among others, of the method of preparing one a
nanostructure includes: providing a nanospecies; and forming a
hydrophobic protection structure around the nanospecies that
includes at least one compound selected from a capping ligand, an
amphiphilic copolymer, and combinations thereof.
An embodiment, among others, of the method of detecting a target in
a subject includes: providing one of the nanostructures described
above having a bio-compatibility compound disposed substantially on
the surface of the hydrophobic protection structure, and at least
one probe disposed substantially on the surface of the hydrophobic
protection structure, wherein a first probe has an affinity for the
target; introducing the nanostructure to a subject; and determining
the presence of the target in the subject corresponding to the
probe by detecting the nanospecies.
An embodiment, among others, of the method of treating a disease in
a subject includes providing one of the nanostructures described
above having a bio-compatibility compound disposed substantially on
the surface of the hydrophobic protection structure, and at least
one probe disposed substantially on the surface of the hydrophobic
protection structure, wherein a first probe has an affinity of the
target; introducing the nanostructure to the subject in need of
treatment of the disease.
BRIEF DESCRIPTION OF THE DRAWINGS
Further aspects of the present disclosure will be more readily
appreciated upon review of the detailed description of its various
embodiments, described below, when taken in conjunction with the
accompanying drawings.
FIG. 1 illustrates an exemplar embodiment of a nanostructure.
FIGS. 2A through 2D illustrates an exemplary method of forming the
nanostructure illustrated in FIG. 1.
FIG. 3A illustrates a schematic of bioconjugated quantum dots for
in vivo cancer targeting and imaging.
FIG. 3B illustrates a chemical modification of a triblock copolymer
with an 8-carbon side chain.
FIG. 3C illustrates the permeation and retention of QD probes via
leaky tumor vasculatures (passive targeting), and high affinity
binding of QD-antibody conjugates to tumor antigens (active
targeting).
FIG. 4 illustrates immunocytochemical studies of QD-PSMA antibody
(Ab) binding activity in cultured prostate cancer cells. The top
panels illustrate bright-field and fluorescence images that were
obtained for PSMA-positive C4-2 cells as revealed by the presence
of QD-PSMA-Ab complex on the cell surface. The middle panels
illustrate negative staining that was detected in C4-2 cells
exposed to QD-PEG in the absence of PSMA Ab. The bottom panels
illustrate negative staining that was noted in PC-3 cells, which
lack PSMA expression.
FIGS. 5A and 5B illustrate a histological examination of QD uptake,
retention, and distribution in six different normal host organs
(FIG. 5A) and in C4-2 tumor (FIG. 5B) xenografts maintained in
athymic nude mice. QD uptake and retention was evaluated by using
three surface modifications as indicated by the left, middle, and
right columns. In the left column the QD is coated with surface
carboxylic acid groups (6.0 nmol and 6 hrs circulation). In the
middle column the QD is surface coated with PEG groups (6.0 nmol
and 24 hrs circulation). In the right column the QD is surface
modified by PEG and bioconjugated with a PSMA antibody (0.4 nmol
and 2 hrs circulation). The left and middle columns are the same
except that the amount of QD injection was all reduced to 0.4 nmol
and the circulation was reduced to 2 hours. All images were
obtained from 5-10 .mu.m-thin tissue sections on an
epi-fluorescence microscope. All the tumors had similar sizes,
measuring about 0.5-1 cm in diameter along the long axis. QDs were
detected by their characteristic red-orange fluorescence, and all
other signals were due to background autofluorescence.
FIGS. 6A through 6D illustrate spectral imaging of QD-PSMA Ab
conjugates in live animals harbored with C4-2 tumor xenografts.
Orange-red fluorescence signals indicate a prostate tumor growing
in a live mouse (FIGS. 6B and 6D). Control studies using a healthy
mouse (no tumor) and the same amount of QD injection showed no
localized fluorescence signals (FIGS. 6A and 6C). FIG. 6A is the
original image; FIG. 6B is an unmixed autofluorescence image; FIG.
6C is an unmixed QD image; and FIG. 6D is a super-imposed image.
After in vivo imaging, histological and immunocytochemical
examinations confirmed that the QD signals came from an underlying
tumor.
FIG. 7 illustrates in vivo fluorescence images of tumor-bearing
mice using QD probes with three different surface modifications:
carboxylic acid groups (left), PEG groups (middle), and PEG-PSMA Ab
conjugates (right). For each surface modification, a color image
(top), two fluorescence spectra from QD and animal skin (middle),
and a spectrally resolved image (bottom) were obtained from the
live mouse models bearing C4-2 human prostate tumors of similar
sizes (0.5-1.0 cm in diameter). The amounts of injected QDs and the
lengths of circulation were: 6 nmol and 6 hours for the COOH probe;
6 nmol and 24 hours for the PEG probe; and 0.4 nmol and 2 hours for
the PSMA probe (same as in FIG. 4). The site of QD injection was
observed as a red spot on the mouse tail. The spectral feature at
about 700 nm (QD curve, middle panel) was an artifact caused by
mathematical fitting of the original QD spectrum, which has little
or no effect on background removal.
FIG. 8A illustrates a sensitivity and spectral comparison between
QD-tagged and GFP-transfected cancer cells, and FIG. 8B illustrates
a simultaneous in vivo imaging of multicolor QD-encoded microbeads.
The right-hand images in FIGS. 8A and 8B show QD-tagged cancer
cells (upper) and GFP-labeled cells (lower).
FIGS. 9A and 9B illustrate a comparison of red-emitting QDs and red
organic dyes for in vivo optical imaging. FIG. 9A illustrates an
image that was obtained with blue excitation at 470 nm and 515 nm
long-pass emission, and FIG. 9B illustrates an image that was
obtained with yellow excitation at 570 nm and 600 nm long-pass
emission. Cancer cells (MDA-MB-231) were labeled with either Tat-QD
or Tat-nanobeads (250-nm particles with embedded organic dyes,
.lamda..sub.ex=575, and .lamda..sub.em=615 nm, Sigma-Aldrich, St
Louis, Mo.) in cell culture. Prior to injection, the QD- and
dye-labeled cells were similarly bright when examined with an
epi-fluorescence microscope. Approximately 1000 cells were injected
subcutaneously into a living mouse at two adjacent sites for in
vivo imaging.
FIG. 10A illustrates a graph depicting autofluorescence spectra of
a nude mouse skin specimen obtained at four excitation wavelengths
(.lamda.=350, 480, 535 and 560 nm). Note the presence of
significant autofluorescence up to 800-850 nm and a background peak
at about 670 nm. FIG. 10B illustrates a comparison of mouse skin
and QD emission spectra obtained under the same excitation
conditions, demonstrating that the QD signals can be shifted to a
spectral region where the autofluorescence is reduced.
DETAILED DESCRIPTION
In accordance with the purpose(s) of the present disclosure, as
embodied and broadly described herein, embodiments of the present
disclosure, in one aspect, relate to bioconjugated nanostructures
(hereinafter nanostructures), methods of fabricating these
nanostructures, and methods of using these nanostructures. The
nanostructures are distinguishable and can be individually
detected. In this regard, the nanostructures can be modified so
that the nanostructures interact with certain target molecules,
which allow detection of the target molecules (e.g., in-vivo)
thereby determining the area in which the target molecules are
located, for example.
The nanostructures can be used in many areas such as, but not
limited to, biomolecule array systems, biosensing, biolabeling,
gene expression studies, protein studies, medical diagnostics,
diagnostic libraries, microfluidic systems, delivery vehicles,
cosmetics, detergents, and nanoparticle-polymer arrays (e.g.,
self-assembly, lithography and patterning). In particular, the
nanostructures can be used in in-vivo diagnostic and/or therapeutic
applications such as, but not limited to, targeting and/or imaging
of diseases and/or conditions (e.g., identify the type of disease,
locate the proximal locations of the disease, and deliver drugs to
the diseased cells (e.g., cancer cells) in living animals, as
described in detail Example 1. The nanostructures in combination
with spectral imaging can be used for multiplexed imaging and
detection of genes, proteins, and the like, in single living
cells.
Embodiments of the nanostructure include, but are not limited to, a
nanospecies (e.g., quantum dots, metal particles and metal oxide
particles) and a hydrophobic protection structure that encapsulates
the nanospecies. In addition, the nanostructure can include, but is
not limited to, a bio-compatibility compound (e.g., polyethylene
glycol (MW about 500 to 50,000 and 1000 to 10,000), dextran, and
derivatives such as amino-dextran and carboxy-dextran, and
polysaccharides) and a probe (e.g., polynucleotide, polypeptide, a
therapeutic agent, and/or a drug). The bio-compatibility compound
and/or the probe are substantially disposed (e.g., attached to the
surface of the hydrophobic protection structure and/or attached
within the hydrophobic protection structure) on the hydrophobic
protection structure. The hydrophobic protection structure includes
a capping ligand and/or a amphiphilic copolymer (e.g., amphiphilic
block copolymers, amphiphilic random copolymers, amphiphilic
alternating copolymers, amphiphilic periodic copolymers, and
combinations thereof).
In another embodiment, the nanostructure can include two or more
nanospecies or two of more types of nanospecies. In addition, the
nanostructure can include a hydrophobic protection structure having
two or more copolymers (e.g., two or more block copolymers).
Further, the nanostructure can include multiple nanospecies and
multiple copolymers (e.g., block copolymers). In addition, the
nanostructure can include two or more different types of probes
having different functions. Furthermore, the nanospecies and the
copolymers (e.g., block copolymers) can be assembled into micro and
macro structures.
In still another embodiment, the nanostructure can be included in a
porous material such as, but is not limited to, a mesoporous
material (e.g., a pore diameter of about 1 to 100 nanometers (nm)),
a macroporous material (e.g., a pore diameter of greater than about
100 nm), and a hybrid mesoporous/macroporous material. The porous
material can be made of a material such as, but not limited to, a
polymer, a copolymer, a metal, a silica material, cellulose,
ceramic, zeolite, and combinations thereof. The preferred porous
materials are silica materials and polystyrene and polystyrene
co-polymers (e.g., divinylbenzene, methacrylic acid, maleic acid).
The shape of the porous material can be, but is not limited to,
spherical, cubic, monolith (i.e., bulk material), two dimensional
and three dimensional arrays. The preferred shape of the porous
material is spherical (e.g., silica beads and polymer beads (e.g.,
chromatographic beads), ceramic, and molecular sieves).
FIG. 1 illustrates an exemplar embodiment of the nanostructure 100.
The nanostructure includes, but is not limited to, a nanospecies
102 having a hydrophobic protection structure 104 that encapsulates
the nanospecies 102. In addition, the nanostructure 100 can
include, but is not limited to, a bio-compatibility compound 112
and a probe 114. The hydrophobic protection structure 104 includes
a capping ligand layer 106 and/or a copolymer layer 108 (e.g.,
amphiphilic block copolymer). The following illustrative examples
will use amphiphilic block copolymers, but other copolymers such
as, but not limited to, amphiphilic random copolymers, amphiphilic
alternating copolymers, amphiphilic periodic copolymers, and
combinations thereof, can be used in combination with block
copolymers, as well as individually or in any combination. In
addition, the term "amphiphilic block copolymer" will be termed
"block copolymer" hereinafter.
In general, the nanostructure 100 can be formed in a manner
described in FIGS. 2A through 2D. FIG. 2A illustrates the
nanospecies 102, while FIG. 2B illustrates the capping ligand 106
disposed on the nanospecies 102. FIG. 2C illustrates the block
copolymer disposed on the capping ligand layer 106 to form the
hydrophobic protection structure 104. FIG. 2D illustrates the
addition of the bio-compatibility compound 112 and the probe 114
onto the hydrophobic protection structure 104.
As mentioned above, the nanostructure can include a number of types
of nanospecies such as, but not limited to, semiconductor, metal,
and metal oxide nanoparticles (e.g., gold, silver, copper,
titanium, nickel, platinum, palladium, oxides thereof (e.g.,
Cr.sub.2O.sub.3, CO.sub.3O.sub.4, NiO, MnO, CoFe.sub.2O.sub.4, and
MnFeO.sub.4), and alloys thereof), metalloid and metalloid oxide
nanoparticles, the lanthanide series metal nanoparticles, and
combinations thereof. In particular, semiconductor quantum dots are
described in more detail below and in U.S. Pat. No. 6,468,808 and
International Patent Application WO 03/003015, which are
incorporated herein by reference. Furthermore, the magnetic
nanoparticles (e.g., those having magnetic or paramagnetic
properties) can include, but are not limited to, iron nanoparticles
and iron composite nanoparticles (e.g., Fe.sub.2O.sub.3,
Fe.sub.3O.sub.4, FePt, FeCo, FeAl, FeCoAl, CoFe.sub.2O.sub.4, and
MnFeO.sub.4).
As indicated above, the nanostructure can include quantum dots such
as, but not limited to, luminescent semiconductor quantum dots. In
general, quantum dots include a core and a cap, however, uncapped
quantum dots can be used as well. The "core" is a nanometer-sized
semiconductor. While any core of the IIA-VIA, IIIA-VA or IVA-IVA,
IVA-VIA semiconductors can be used in the context of the present
disclosure, the core must be such that, upon combination with a
cap, a luminescent quantum dot results. A IIA-VIA semiconductor is
a compound that contains at least one element from Group IIB and at
least one element from Group VIA of the periodic table, and so on.
The core can include two or more elements. In one embodiment, the
core is a IIA-VIA, IIIA-VA or IVA-IVA semiconductor that ranges in
size from about 1 nm to about 20 nm. In another embodiment, the
core is more preferably a IIA-VIA semiconductor and ranges in size
from about 2 nm to about 10 nm. For example, the core can be CdS,
CdSe, CdTe, ZnSe, ZnS, PbS, PbSe or an alloy.
The "cap" is a semiconductor that differs from the semiconductor of
the core and binds to the core, thereby forming a surface layer on
the core. The cap can be such that, upon combination with a given
semiconductor core a luminescent quantum dot results. The cap
should passivate the core by having a higher band gap than the
core. In one embodiment, the cap is a IIA-VIA semiconductor of high
band gap. For example, the cap can be ZnS or CdS. Combinations of
the core and cap can include, but are not limited to, the cap is
ZnS when the core is CdSe or CdS, and the cap is CdS when the core
is CdSe. Other exemplary quantum dots include, but are not limited
to, CdS, ZnSe, CdSe, CdTe, CdSe.sub.xTe.sub.1-x, InAs, InP, PbTe,
PbSe, PbS, HgS, HgSe, HgTe, CdHgTe, and GaAs.
The wavelength emitted (i.e., color) by the quantum dots can be
selected according to the physical properties of the quantum dots,
such as the size and the material of the nanocrystal. Quantum dots
are known to emit light from about 300 nanometers (nm) to 1700 nm
(e.g., UV, near IR, and IR). The colors of the quantum dots
include, but are not limited to, red, blue, green, and combinations
thereof. The color or the fluorescence emission wavelength can be
tuned continuously. The wavelength band of light emitted by the
quantum dot is determined by either the size of the core or the
size of the core and cap, depending on the materials which make up
the core and cap. The emission wavelength band can be tuned by
varying the composition and the size of the QD and/or adding one or
more caps around the core in the form of concentric shells.
The intensity of the color of the quantum dots can be controlled.
For each color, the use of 10 intensity levels (0, 1, 2, . . . 9)
gives 9 unique codes (10.sup.1-1), because level "0" cannot be
differentiated from the background. The number of codes increase
exponentially for each intensity and each color used. For example,
a three color and 10 intensity scheme yields 999 (10.sup.3-1)
codes, while a six color and 10 intensity scheme has a theoretical
coding capacity of about 1 million (10.sup.6-1). In general, n
intensity levels with m colors generate (n.sup.m-1) unique codes.
Use of the intensity of the quantum dots has applications in
nanostructures including a plurality of different types of quantum
dots having different intensity levels and also in nanostructures
including a plurality of different types of quantum dots having
different intensity levels that are included in a porous material.
The quantum dots are capable of absorbing energy from, for example,
an electromagnetic radiation source (of either broad or narrow
bandwidth), and are capable of emitting detectable electromagnetic
radiation at a narrow wavelength band when excited. The quantum
dots can emit radiation within a narrow wavelength band (FWHM, full
width at half maximum) of about 40 nm or less, thus permitting the
simultaneous use of a plurality of differently colored quantum dots
with little or no spectral overlap.
The synthesis of quantum dots is well known and is described in
U.S. Pat. Nos. 5,906,670; 5,888,885; 5,229,320; 5,482,890;
6,468,808; 6,306,736; 6,225,198, etc., International Patent
Application WO 03/003015, (all of which are incorporated herein by
reference) and in many research articles. The wavelengths emitted
by quantum dots and other physical and chemical characteristics
have been described in U.S. Pat. No. 6,468,808 and International
Patent Application WO 03/003015 and will not be described in any
further detail. In addition, methods of preparation of quantum dots
are described in U.S. Pat. No. 6,468,808 and International Patent
Application WO 03/003015 and will not be described any further
detail.
As mentioned above, the hydrophobic protection structure includes
the capping ligand and/or the block copolymer. In particular, when
the nanospecies is a quantum dot, the hydrophobic protection layer
includes the capping ligand and the block copolymer, where the
capping ligand and the block copolymer interact with one another to
form the hydrophobic protection structure. As such, the capping
ligand and the block copolymer are selected to form an appropriate
hydrophobic protection structure. For example, the block copolymer
and the nanospecies can interact through interactions such as, but
not limited to, hydrophobic interactions, hydrophilic interactions,
pi-stacking, etc., depending on the surface coating of the
nanospecies and the molecular structure of polymers. Additional
details regarding the capping ligand and the block copolymer are
provided in Example 1 below.
The capping ligand caps the nanospecies (e.g., quantum dot) and
forms a layer on the nanospecies, which subsequently bonds with the
block copolymer to form the hydrophobic protection structure. The
capping ligand can include compounds such as, but not limited to,
an O.dbd.PR.sub.3 compound, an O.dbd.PHR.sub.2 compound, an
O.dbd.PHR.sub.1 compound, a H.sub.2NR compound, a HNR.sub.2
compound, a NR.sub.3 compound, a HSR compound, a SR.sub.2 compound,
and combinations thereof. "R" can be a C.sub.1 to C.sub.18
hydrocarbon, such as but not limited to, linear hydrocarbons,
branched hydrocarbons, cyclic hydrocarbons, substituted
hydrocarbons (e.g., halogenated), saturated hydrocarbons,
unsaturated hydrocarbons, and combinations thereof. Preferably, the
hydrocarbon is a saturated linear C.sub.4 to C.sub.18 hydrocarbon,
a saturated linear C.sub.6 to C.sub.18 hydrocarbon, and a saturated
linear C.sub.18 hydrocarbon. A combination of R groups can be
attached to P, N, or S. In particular, the chemical can be selected
from tri-octylphosphine oxide, stearic acid, and octyldecyl
amine.
As mentioned above, the copolymer includes, but is not limited to,
amphiphilic block copolymers, amphiphilic random copolymers,
amphiphilic alternating copolymers, amphiphilic periodic
copolymers, and combinations thereof. The amphiphilic random
copolymer can include, but is not limited to random copolymer
poly(methyl acrylate-co-acrylic acid); random copolymer poly(methyl
methacrylate-co-n-butyl acrylate); random copolymer poly(methyl
methacrylate-co-hydroxypropyl acrylate); random copolymer
poly(styrene-co-p-carboxyl chloro styrene); random copolymer
poly(styrene-co-4-hydroxystyrene); random copolymer
poly(styrene-co-4-vinyl benzoic acid); random copolymer
poly(styrene-co-4-vinyl pyridine); (and combinations thereof. The
amphiphilic alternating copolymer can include, but is not limited
to, poly(maleic anhydride-alt-1-octadecene), poly(maleic
anhydride-alt-1-tetradecene), alternating copolymer poly(carbo
tert.butoxy .alpha.-methyl styrene-alt-maleic anhydride) and
alternating copolymer poly(carbo tert.butoxy norbornene-alt-maleic
anhydride), and combinations thereof.
The block copolymer includes amphiphilic di- and or triblock
copolymers. In addition, the copolymer can include hydrocarbon side
chains such as, but not limited to, 1-18-carbon aliphatic side
chains, 1-18-carbon alkyl side chains, and combinations thereof.
Furthermore, the di or tri block copolymers have at least one
hydrophobic block and at least one hydrophilic block.
The following is in an exemplary list of amphiphilic random and
alternating copolymers: random copolymer poly(dimethyl
siloxane-co-diphenyl siloxane); random copolymer poly(methyl
acrylate-co-acrylic acid); random copolymer poly(methyl
methacrylate-co-n-butyl acrylate); random copolymer poly(methyl
methacrylate-co-t-butyl acrylate); random copolymer poly(methyl
methacrylate-co-hydroxypropyl acrylate); random copolymer
poly(tetrahydrofuranyl methacrylate-co-ethyl methacrylate); random
copolymer poly(styrene-co-4-bromostyrene); random copolymer
poly(styrene-co-butadiene); random copolymer
poly(styrene-co-diphenyl ethylene); random copolymer
poly(styrene-co-t-butyl methacrylate); random copolymer
poly(styrene-co-t-butyl-4-vinyl benzoate); random copolymer
poly(styrene-co-p-carboxyl chloro styrene); random copolymer
poly(styrene-co-p-chloromethyl styrene); random copolymer
poly(styrene-co-methyl methacrylate); random copolymer
poly(styrene-co-4-hydroxystyrene); random copolymer
poly(styrene-co-4-vinyl benzoic acid); random copolymer
poly(styrene-co-4-vinyl pyridine); alternating copolymer poly(carbo
tert.butoxy .alpha.-methyl styrene-alt-maleic anhydride);
alternating copolymer poly(carbo tert.butoxy norbornene-alt-maleic
anhydride); alternating copolymer poly(.alpha.-methyl
styrene-alt-methyl methacrylate); and alternating copolymer
poly(styrene-alt-methyl methacrylate).
The following in an exemplary list of amphiphilic copolymers:
poly((meth)acrylic acid) based copolymers (e.g., poly(acrylic
acid-b-methyl methacrylate); poly(methyl methacrylate-b-acrylic
acid); poly(methyl methacrylate-b-sodium acrylate); poly(sodium
acrylate-b-methyl methacrylate); poly(methacrylic acid-b-neopentyl
methacrylate); poly(neopentyl methacrylate-b-methacrylic acid);
poly(t-butyl methacrylate-b-ethylene oxide); poly(methyl
methacrylate-b-sodium methacrylate); and poly(methyl
methacrylate-b-N,N-dimethyl acrylamide)), polydiene and
hydrogenated polydiene based copolymers (e.g., poly(butadiene(1,2
addition)-b-methylacrylic acid; poly(butadiene(1,4
addition)-b-acrylic acid); poly(butadiene(1,4 addition)-b-sodium
acrylate); poly(butadiene(1,4 addition)-b-ethylene oxide;
poly(butadiene(1,2 addition)-b-ethylene oxide); poly(butadiene(1,2
addition)-b-ethylene oxide)-hydroxy benzoic ester terminal group;
4-methoxy benzyolester terminated poly(butadiene-b-ethylene oxide)
diblock copolymer; poly(butadiene-b-N-methyl 4-vinyl pyridinium
iodide); poly(isoprene-b-N-methyl 2-vinyl pyridinium iodide);
poly(isoprene-b-ethylene oxide) (1,4 addition);
poly(isoprene-b-ethylene oxide) (1,2 and 3,4 addition);
poly(propylene-ethylene-b-ethylene oxide); and hydrogonated
poly(isoprene-b-ethylene oxide) (1,2 addition)), hydrogentated
diene based copolymers (e.g., poly(ethylene-b-ethylene oxide) and
poly(isoprene-b-ethylene oxide)), poly(ethylene oxide) based
copolymers (e.g., poly(ethylene oxide-b-acrylic acid);
poly(ethylene oxide-b-.di-elect cons.-caprolactone); poly(ethylene
oxide-b-6-(4'-cyanobiphenyl-4-yloxy)hexyl methacrylate);
poly(ethylene oxide-b-lactide); poly(ethylene
oxide-b-2-hydroxyethyl methacrylate); poly(ethylene oxide-b-methyl
methacrylate); poly(-methyl methacrylate-b-ethylene oxide);
poly(ethylene oxide-b-methacrylic acid); poly(ethylene
oxide-b-2-methyl oxazoline); poly(ethylene oxide-b-propylene
oxide); poly(ethylene oxide-b-t-butyl acrylate); poly(ethylene
oxide-b-tetrahydrofurfuryl methacrylate); and poly(ethylene
oxide-b-N,N-dimethylethylmethacrylate)), polyisobutylene based
copolymers (e.g., poly(isobutylene-b-ethylene oxide)), polystyrene
based copolymers (e.g., poly(styrene-b-acrylic acid);
poly(styrene-b-sodium acrylate); poly(styrene-b-acrylamide);
poly(p-chloromethyl styrene-b-acrylamide);
poly(styrene-co-p-chloromethyl styrene-b-acrylamide);
poly(styrene-co-p-chloromethyl styrene-b-acrylic acid);
poly(styrene-b-cesium acrylate); poly(styrene-b-ethylene oxide);
poly(4-styrenesulfonic acid-b-ethylene oxide);
poly(styrene-b-methacrylic acid); poly(styrene-b-sodium
methacrylate); poly(styrene-b-N-methyl 2-vinyl pyridinium iodide);
and poly(styrene-b-N-methyl-4-vinyl pyridinium iodide)),
polysiloxane based copolymers (e.g.,
poly(dimethylsiloxane-b-acrylic acid)), poly(2-vinyl naphthalene)
based copolymers (e.g., poly(2-vinyl naphthalene-b-acrylic acid)),
poly (vinyl pyridine and N-methyl vinyl pyridinium iodide) based
copolymers (e.g., poly(2-vinyl pyridine-b-ethylene oxide);
poly(N-methyl 2-vinyl pyridinium iodide-b-ethylene oxide); and
poly(N-methyl 4-vinyl pyridinium iodide-b-methyl
methacrylate)).
The following in an exemplary list of amphiphilic diblock
copolymers: poly(meth)acrylate based copolymers (e.g., poly(n-butyl
acrylate-b-methyl methacrylate); poly(n-butyl
acrylate-b-dimethylsiloxane-co-diphenyl siloxane); poly(t-butyl
acrylate-b-methyl methacrylate); poly(t-butyl
acrylate-b-4-vinylpyridine); poly(2-ethyl hexyl acrylate-b-4-vinyl
pyridine); poly(t-butyl methacrylate-b-2-vinyl pyridine);
poly(2-hydroxyl ethyl acrylate-b-neopentyl acrylate);
poly(2-hydroxyl ethyl methacrylate-b-neopentyl methacrylate);
poly(2-hydroxyl ethyl methacrylate-b-n-butyl methacrylate);
poly(2-hydroxyl ethyl methacrylate-b-t-butyl methacrylate);
poly(methyl methacrylate-b-acrylonitrile); poly(methyl
methacrylate-b-t-butyl methacrylate); poly(isotactic methyl
methacrylate-b-syndiotactic methyl methacrylate); poly(methyl
methacrylate-b-t-butyl acrylate); poly(methyl
methacrylate-b-trifluroethyl methacrylate); poly(methyl
methacrylate-b-2-hydroxyethyl methacrylate with cholesteryl
chloroformate); poly(methyl methacrylate-b-disperse red 1
acrylate); poly(methyl methacrylate-b-2-hydroxyethyl methacrylate);
poly(methyl methacrylate-b-neopentyl acrylate); and
poly(methacrylate-b-2-pyranoxy ethyl methacrylate)), polydiene
based copolymers (e.g., poly(butadiene(1,2 addition)-b-i-butyl
methacrylate); poly(butadiene(1,2 addition)-b-s-butyl
methacrylate); poly(butadiene(1,4 addition)-b-t-butyl acrylate;
poly(butadiene(1,2 addition)-b-t-butyl acrylate; poly(butadiene(1,2
addition)-b-t-butyl methacrylate); poly(butadiene(1,4
addition)-b-.di-elect cons.-caprolactone); poly(butadiene((1,4
addition)-b-dimethylsiloxane); poly(butadiene(1,4
addition)-b-methyl methacrylate) (syndiotactic); poly(butadiene(1,2
addition)-b-methyl methacrylate); poly(butadiene(1,4
addition)-b-4-vinyl pyridine; poly(isoprene(1,4 addition)-b-methyl
methacrylate(syndiotactic)); poly(isoprene(1,4 addition)-b-2-vinyl
pyridine; poly(isoprene(1,2 addition)-b-4-vinyl pyridine); and
poly(isoprene(1,4 addition)-b-4-vinyl pyridine)), polyisobutylene
based copolymers (e.g., poly(isobutylene-b-t-butyl methacrylate);
poly(isobutylene-b-.di-elect cons.-caprolactone);
poly(isobutylene-b-dimethylsiloxane); poly(isobutylene-b-methyl
methacrylate); poly(isobutylene-b-4-vinyl pyridine), polystyrene
based copolymers (e.g., poly(styrene-b-n-butyl acrylate);
poly(styrene-b-t-butyl acrylate); poly(styrene-b-t-butyl acrylate),
broad distribution; poly(styrene-b-disperse red 1 acrylate);
poly(p-chloromethyl styrene-b-t-butyl acrylate);
poly(styrene-b-N-isopropyl acrylamide); poly(styrene-b-n-butyl
methacrylate); poly(styrene-b-t-butyl methacrylate);
poly(styrene-b-cyclohexyl methacrylate);
poly(styrene-b-2-cholesteryloxycarbonyloxy ethyl methacrylate);
poly(styrene-b-N,N-dimethyl amino ethyl methacrylate);
poly(styrene-b-ethyl methacrylate); poly(styrene-b-2-hydroxyethyl
methacrylate); poly(styrene-b-2-hydroxypropyl methacrylate);
poly(styrene-b-methyl methacrylate);
poly(styrene-b-methylmethacrylate); poly(styrene-b-n-propyl
methacrylate); poly(styrene-b-butadiene(1,4 addition));
poly(styrene-b-butadiene(1,2 addition));
poly(styrene-b-isoprene(1,4 addition)); poly(styrene-b-isoprene(1,2
addition or 3,4 addition)); poly(styrene-b-isoprene(1,4 addition)),
hydrogenated; tapered block copolymer poly(styrene-b-butadiene);
tapered block copolymer poly(styrene-b-ethylene);
poly(styrene-b-.di-elect cons.-caprolactone);
poly(styrene-b-1-lactide); poly(styrene-b-dimethylsiloxane),
trimethylsilane endgroup; poly(styrene-b-dimethylsiloxane), silanol
endgroup; poly(styrene-b-methyl phenyl siloxane);
poly(styrene-b-ferrocenyldimethylsilane); poly(styrene-b-t-butyl
styrene); poly(styrene-b-t-butoxystyrene);
poly(styrene-b-4-hydroxyl styrene); poly(4-amino benzyl-b-styrene);
poly(styrene-b-2-vinyl pyridine); poly(styrene-b-4-vinyl pyridine);
and poly(.alpha.-methylstyrene-b-4-vinyl pyridine), poly(vinyl
naphthalene) based copolymers (e.g., poly(2-vinyl
naphthalene-b-n-butyl acrylate), poly(2-vinyl naphthalene-b-t-butyl
acrylate); poly(2-vinyl naphthalene-b-methyl methacrylate); and
poly(2-vinyl naphthalene-b-dimethylsiloxane)), poly(vinyl pyridine)
based copolymers (e.g., poly(2-vinyl pyridine-b-.di-elect
cons.-caprolactone); poly(2-vinyl pyridine-b-methyl methacrylate);
and poly(4-vinyl pyridine-b-methyl methacrylate)), poly (propylene
oxide-b-.di-elect cons.-caprolactone) (e.g., poly (propylene
oxide-b-F-caprolactone)), polysiloxane based copolymers (e.g.,
poly(dimethylsiloxane-b-n-butyl acrylate);
poly(dimethylsiloxane-b-t-butyl acrylate);
poly(dimethylsiloxane-b-t-butyl methacrylate);
poly(dimethylsiloxane-b-.di-elect cons.-caprolactone);
poly(dimethylsiloxane-b-6-(4'-cyanobiphenyl-4-yloxy)hexyl
methacrylate); poly(dimethylsiloxane-b-1-ethoxy ethyl
methacrylate); poly(dimethylsiloxane-b-hydroxy ethyl acrylate); and
poly(dimethylsiloxane-b-methyl methacrylate)), adipic anhydride
based copolymers (e.g., poly(ethylene oxide-b-adipic anhydride);
poly(propylene oxide-b-adipic anhydride); poly(dimethyl
siloxane-b-adipic anhydride); poly(methyl methacrylate-b-adipic
anhydride); and poly(2-vinyl pyridine-b-adipic anhydride)).
The following in an exemplary list of amphiphilic a-b-a triblock
copolymers: poly((meth)acrylate) based triblock copolymers (e.g.,
poly(n-butyl acrylate-b-9,9-di-n-hexyl-2,7-fluorene-b-n-butyl
acrylate); poly(t-butyl
acrylate-b-9,9-di-n-hexyl-2,7-fluorene-b-t-butyl acrylate);
poly(acrylic acid-b-9,9-di-n-hexyl-2,7-fluorene-b-acrylic acid);
poly(t-butyl acrylate-b-methyl methacrylate-b-t-butyl acrylate);
poly(t-butyl acrylate-b-styrene-b-t-butyl acrylate); poly(methyl
methacrylate-b-butadiene(1,4 addition)-b-methyl methacrylate);
poly(methyl methacrylate-b-n-butyl acrylate-b-methyl methacrylate);
poly(methyl methacrylate-b-t-butyl acrylate-b-methyl methacrylate);
poly(methyl methacrylate-b-t-butyl methacrylate acid-b-methyl
methacrylate); poly(methyl methacrylate-b-methacrylic acid-b-methyl
methacrylate); poly(methyl methacrylate-b-dimethylsiloxane-b-methyl
methacrylate); poly(methyl
methacrylate-b-9,9-di-n-hexyl-2,7-fluorene-b-methyl methacrylate);
poly(methyl methacrylate-b-styrene-b-methyl methacrylate);
poly(trimethylamonium iodide ethyl
methacrylate-b-9,9-di-n-hexyl-2,7-fluorene-b-trimethylamonium
iodide ethyl methacrylate); poly(N,N-dimethyl amino ethyl
methacrylate-b-9,9-di-n-hexyl-2,7-fluorene-b-N,N-dimethyl amino
ethyl methacrylate); and poly(N,N-dimethyl amino ethyl
methacrylate-b-propylene oxide-b-N,N-dimethyl amino ethyl
methacrylate)), polybutadiene based triblock copolymers (e.g.,
poly(butadiene(1,4 addition)-b-styrene-b-butadiene(1,4 addition))),
poly(oxirane) based triblock copolymers (e.g., poly(ethylene
oxide-b-9,9-di-n-hexyl-2,7-fluorene-b-ethylene oxide);
poly(ethylene oxide-b-propylene oxide-b-ethylene oxide);
poly(ethylene oxide-b-styrene-b-ethylene oxide); and poly(propylene
oxide-b-dimethyl siloxane-b-propylene oxide)), polylactone and
polylactide diblock copolymers (e.g., poly(lactide-b-ethylene
oxide-b-lactide); poly(caprolactone-b-ethylene
oxide-b-caprolactone); and alpha,-.omega. diacrylonyl terminated
poly(lactide-b-ethylene oxide-b-lactide)), polyoxazoline based
triblock copolymers (e.g., poly(2-methyl oxazoline-b-dimethyl
siloxane-b-2-methyl oxazoline))), polystyrene based triblock
copolymers (e.g., poly(styrene-b-acrylic acid-b-styrene);
poly(styrene-b-butadiene (1,4 addition)-b-styrene);
poly(styrene-b-butadiene (1,2 addition)-b-styrene);
poly(styrene-b-butylene-b-styrene); poly(styrene-b-n-butyl
acrylate-b-styrene); poly(styrene-b-t-butyl acrylate-b-styrene);
poly(styrene-b-9,9-di-n-hexyl-2,7-fluorene-b-styrene);
poly(styrene-b-ethyl acrylate-b-styrene);
poly(styrene-b-isoprene-b-styrene); poly(styrene-b-ethylene
oxide-b-styrene); poly(styrene-b-4-vinyl pyridine-b-styrene); and
poly(styrene-b-dimethyl siloxane-b-styrene)), poly(vinyl pyridine)
based triblock copolymers (e.g., poly(2-vinyl
pyridine-b-butadiene(1,2 addition)-b-2-vinyl pyridine);
poly(2-vinyl pyridine-b-styrene-b-2-vinyl pyridine); and
poly(4-vinyl pyridine-b-styrene-b-4-vinyl pyridine).
The following in an exemplary list of amphiphilic a-b-c triblock
copolymers: poly(styrene-b-butadiene-b-methyl methacrylate) (e.g.,
poly(styrene-b-butadiene-b-methyl methacrylate)),
poly(styrene-b-butadiene-b-2-vinyl pyridine) (e.g.,
poly(styrene-b-butadiene-b-2-vinyl pyridine)),
poly(styrene-b-t-butyl acrylate-b-methyl methacrylate) (e.g.,
poly(styrene-b-t-butyl acrylate-b-methyl methacrylate)),
poly(styrene-b-isoprene-b-glycidyl methacrylate) (e.g.,
poly(styrene-b-isoprene-b-glycidyl methacrylate)),
poly(styrene-b-2-vinyl pyridine-b-ethylene oxide) (e.g.,
poly(styrene-b-2-vinyl pyridine-b-ethylene oxide)),
poly(styrene-b-anthracene methyl methacrylate-b-methymethacrylate)
(e.g., poly(styrene-b-anthracene methyl
methacrylate-b-methymethacrylate)), poly(styrene-b-t-butyl
acrylate-b-2-vinyl pyridine) (e.g., poly(styrene-b-t-butyl
acrylate-b-2-vinyl pyridine)), and poly(styrene-b-t-butyl
methacrylate-b-2-vinyl pyridine) (e.g., poly(styrene-b-t-butyl
methacrylate-b-2-vinyl pyridine)).
The following in an exemplary list of amphiphilic functionalized
diblock and triblock copolymers: amino terminated
poly(dimethylsiloxane-b-diphenylsiloxane); amino terminated
poly(styrene-b-isoprene); amino terminated poly(ethylene
oxide-b-lactone); hydroxy terminated poly(styrene-b-2-vinyl
pyridine); hydroxy terminated polystyrene-b-poly(methyl
methacrylate); .alpha.-hydroxy terminated
poly(styrene-b-butadiene(1,2-addition)); 4-methoxy benzyolester
terminated poly(butadiene-b-ethylene oxide) diblock copolymer;
succinic acid terminated poly(butadiene-b-ethylene oxide) diblock
copolymer; .alpha.,.omega.-disuccinimidyl succinate terminated
poly(ethylene oxide-propylene oxide-ethylene oxide); cabinol at the
junction of poly(styrene-b-isoprene(1,4 addition)); and silane at
the junction of poly(styrene-b-2-vinyl pyridine).
In addition, the following is an exemplary list of amphiphilic
block copolymers: poly(1-vinylpyrrolidone-co-vinyl acetate);
poly(ethylene-co-propylene-co-5-methylene-2-norbornene);
poly(styrene-co-acrylonitrile); poly(2-vinylpyridine-co-styrene);
poly(ethylene-co-methacrylic acid) sodium salt;
poly(acrylonitrile-co-butadiene-co-styrene); poly(vinyl
chloride-co-vinyl acetate-co-maleic acid); poly(ethylene-co-vinyl
acetate); poly(ethylene-co-ethyl acrylate);
poly(4-vinylpyridine-co-styrene); poly(vinyl butyral-co-vinyl
alcohol-co-vinyl acetate); poly(methyl methacrylate co-methacrylic
acid); poly-(vinyl chloride-co-vinyl acetate-co-hydroxypropyl
acrylate); Luviquat.RTM.HM 552; poly(vinyl chloride-co-vinyl
acetate-co-vinyl alcohol); poly(styrene-co-divinylbenzene);
poly(DL-lactide-co-glycolide); poly(acrylonitrile-co-methyl
acrylate); poly[(vinyl chloride-co-(1-methyl-4-vinylpiperazine)];
poly(2-isopropenyl-2-oxazoline-co-methyl methacrylate);
poly(tetrafluoroethylene oxide-co-difluoromethylene oxide)
.alpha.,.omega.-diol, ethoxylated;
poly[dimethylsiloxane-co-methyl(3-hydroxypropyl)siloxane]-graft-poly(ethy-
lene glycol) methyl ether;
poly(acrylonitrile-co-methacrylonitrile);
poly(ethylene-co-1-butene); poly(vinylidene fluoride
co-hexafluoropropylene); poly(ethylene-co-1-octene);
poly(ethylene-co-methyl acrylate);
poly(acrylonitrile-co-butadiene), amine terminated;
poly(perfluoropropylene oxide-co-perfluoroformaldehyde); poly(butyl
methacrylate-co-isobutyl methacrylate); poly(styrene-co-maleic
anhydride), partial isooctyl ester, cumene terminated;
poly(acrylonitrile-co-butadiene-co-acrylic acid), dicarboxy
terminated; poly(vinyl alcohol-co-ethylene);
poly(dimethylsiloxane-co-methylphenylsiloxane);
poly(styrene-co-maleic anhydride); poly(Bisphenol
A-co-epichlorohydrin); poly(styrene-co-butadiene);
poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyvaleric acid];
poly(vinyl alcohol-co-vinyl acetate-co-itaconic acid);
poly(methylstyrene-co-indene), hydrogenated;
poly(4-vinylphenol-co-2-hydroxyethyl methacrylate);
poly(styrene-co-maleic anhydride), cumene terminated; poly(methyl
methacrylate-co-ethylene glycol dimethacrylate);
poly(ethylene-co-propylene); poly(styrene-co-maleic acid), partial
isobutyl/methyl mixed ester; poly(Bisphenol A-co-epichlorohydrin),
glycidyl end-capped; poly(methyl methacrylate-co-methacrylic acid);
poly(2-acrylamido-2-methyl-1-propanesulfonic
acid-co-acrylonitrile); poly(propylene-co-tetrafluoroethylene);
poly(butyl methacrylate-co-methyl methacrylate);
poly(dimethylsiloxane-co-alkylmethylsiloxane); poly(acrylic
acid-co-acrylamide) potassium salt;
poly(oxymethylene-co-1,3-dioxepane);
poly(chlorotrifluoroethylene-co-vinylidene fluoride);
poly(melamine-co-formaldehyde), acrylated solution;
poly(pentafluorostyrene-co-glycidyl methacrylate);
poly(1,1,1,3,3,3-hexafluoroisopropyl methacrylate-co-glycidyl
methacrylate); poly(2,2,3,4,4,4,-hexafluorobutyl
methacrylate-co-glycidyl methacrylate);
poly(2,2,3,3,3-pentafluoropropyl methacrylate-co-glycidyl
methacrylate);
poly[(propylmethacryl-heptaisobutyl-PSS)-co-(n-butylmethacrylate)];
poly(pyromellitic dianhydride-co-4,4'-oxydianiline), amic acid
solution; poly(tert-butyl methacrylate-co-glycidyl methacrylate);
poly[(propylmethacryl-heptaisobutyl-PSS)-co-hydroxyethylmethacrylate];
poly[(m-phenylenevinylene)-co-(2,5-dioctoxy-p-phenylenevinylene)];
poly[(methylmethacrylate)-co-(9-anthracenylmethyl methacrylate)];
poly[(methylmethacrylate)-co-(2-naphthylacrylate)];
poly[methylmethacrylate-co-(7-(4-trifluoromethyl)coumarin
methacrylamide)]; poly[(methylmethacrylate)-co-(9-anthracenylmethyl
acrylate)];
poly[(methylmethacrylate)-co-(9H-carbazole-9-ethylmethacrylate)];
poly[(propylmethacryl-heptaisobutyl-PSS)-co-(methylmethacrylate)];
poly[(isobutylene-alt-maleic acid), ammonium
salt)-co-(isobutylene-alt-maleic anhydride)];
poly(ethylenecarbonyl-co-propylenecarbonyl);
poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethy-
lene]; poly(dimethylsiloxane-co-diphenylsiloxane), trimethylsilyl
terminated; poly(dimethylsiloxane-co-methylhydrosiloxane),
trimethylsilyl terminated;
poly(dimethylsiloxane-co-diphenylsiloxane), divinyl terminated;
poly(styrene-co-methyl methacrylate);
poly(styrene-co-.alpha.-methylstyrene);
poly(1,4-cyclohexanedimethylene terephthalate-co-ethylene
terephthalate); Amberjet.TM. 4200;
poly[dimethylsiloxane-co-methyl(3-hydroxypropyl)siloxane]-graft-poly(ethy-
lene glycol) [3-(trimethylammonio)propyl chloride]ether solution;
poly[dimethylsiloxane-co-methyl(3-hydroxypropyl)siloxane]-graft-poly(ethy-
lene/propylene glycol); poly(ethylene-co-butyl acrylate);
poly(ethylene-co-ethyl acrylate-co-maleic anhydride); poly(ethyl
methacrylate-co-methyl acrylate);
poly(ethylene-co-1-butene-co-1-hexene);
poly(melamine-co-formaldehyde), isobutylated solution;
poly[Bisphenol A carbonate-co-4,4'-(3,3,5-trimethylcyclohexylidene)
diphenol carbonate]; poly(acrylamide-co-acrylic acid);
poly(styrene-co-maleic acid), partial sec-butyl/methyl mixed ester;
poly(4-hydroxybenzoic acid-co-6-hydroxy-2-naphthoic acid);
poly[butylene terephthalate-co-poly(alkylene glycol)
terephthalate]; poly(ethylene-co-vinyl acetate-co-methacrylic
acid); poly(melamine-co-formaldehyde), methylated;
poly(acrylonitrile-co-butadiene), dicarboxy terminated; poly(vinyl
chloride-co-vinyl acetate-co-2-hydroxypropyl acrylate);
poly(tetrafluoroethylene oxide-co-difluoromethylene oxide)
.alpha.,.omega.-diol; poly(melamine-co-formaldehyde), butylated
solution; poly[(phenyl glycidyl ether)-co-formaldehyde];
poly(acrylamide-co-diallyldimethylammonium chloride) solution;
poly(tetrafluoroethylene-co-perfluoro(propylvinyl ether));
poly(4-vinylpyridine-co-butyl methacrylate); poly(dimer
acid-co-alkyl polyamine);
poly(1-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate),
quaternized solution; poly(methyl methacrylate-co-ethyl acrylate);
Luviquat.RTM. FC 550; poly(vinyltoluene-co-.alpha.-methylstyrene);
poly(epichlorohydrin-co-ethylene oxide-co-allyl glycidyl ether);
poly(dimethylsiloxane-co-methylhydrosiloxane);
polybutadiene-graft-poly(methyl acrylate-co-acrylonitrile);
poly(styrene-co-maleic anhydride), partial 2-butoxyethyl ester,
cumene terminated; poly(dimethylamine-co-epichlorohydrin) solution;
poly(ethylene-co-acrylic acid); poly(acrylamide-co-acrylic acid)
partial sodium salt; poly(hexafluoropropylene
oxide-co-difluoromethylene oxide) monoalkylamide;
poly(1-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate)
solution; poly(acrylic acid-co-maleic acid) sodium salt;
poly(ethylene-co-acrylic acid, zinc salt);
poly(ethylene-co-tetrafluoroethylene); poly(2,2,2-trifluoroethyl
methacrylate-co-glycidyl methacrylate); poly(pentabromophenyl
acrylate-co-glycidyl methacrylate);
poly(2,2,3,3,4,4,4-heptafluorobutyl methacrylate-co-glycidyl
methacrylate; poly[methylmethacrylate-co-(disperse yellow 7
methacrylate)]; poly(2,2,3,3-tetrafluoropropyl
methacrylate-co-glycidyl methacrylate); poly(pentabromophenyl
methacrylate-co-glycidylmethacrylate);
poly[methylmethacrylate-co-(Disperse Orange 3 methacrylamide)];
poly[((S)-(
)-1-(4-nitrophenyl)-2-pyrrolidinemethyl)acrylate-co-methylmethacrylate];
poly[(methylmethacrylate)-co-(Disperse Red 13 methacrylate)];
poly[(methylmethacrylate)-co-(Disperse Red 13 acrylate)];
poly[methylmethacrylate-co-(N-(1-naphthyl)-N-phenylacrylamide)];
poly[(propylmethacryl-heptaisobutyl-PSS)-co-styrene];
poly(pyromellitic dianhydride-co-thionin); poly(ethylene
glycol)-co-4-benzyloxybenzyl alcohol, polymer-bound;
poly[(isobutylene-alt-maleimide)-co-(isobutylene-alt-maleic
anhydride)];
poly[dimethylsiloxane-co-(3-aminopropyl)methylsiloxane];
poly[dimethylsiloxane-co-[3-(2-(2-hydroxyethoxy)ethoxy)propyl)methylsilox-
ane]; poly(vinylidene chloride-co-acrylonitrile-co-methyl
methacrylate); poly(ethylene-co-1,2-butylene)diol;
poly(DL-lactide-co-caprolactone) (40:60); poly(methyl
methacrylate-co-butyl methacrylate); poly(tetrafluoroethylene
oxide-co-difluoromethylene oxide).alpha.,.omega.-diol
bis(2,3-dihydroxypropyl ether);
poly[dimethylsiloxane-co-(2-(3,4-epoxycyclohexyl)ethyl)methylsiloxane];
poly(vinyl chloride-co-isobutyl vinyl ether);
poly(indene-co-coumarone);
poly(styrene-co-4-bromostyrene-co-divinylbenzene);
poly(ethylene-co-butyl acrylate-co-carbon monoxide); poly(vinyl
acetate-co-butyl maleate-co-isobornyl acrylate) solution;
poly(3,3',4,4'-benzophenonetetracarboxylic
dianhydride-co-4,4'-oxydianiline/1,3-phenylenediamine), amic acid
(solution); poly(tetrafluoroethylene-co-vinylidene
fluoride-co-propylene); poly(ethylene-co-methacrylic acid) lithium
salt; poly(styrene-co-butadiene-co-methyl methacrylate);
poly(vinylidene chloride-co-vinyl chloride); poly(styrene-co-maleic
acid), partial isobutyl ester; poly[4,4'-methylenebis(phenyl
isocyanate)-alt-1,4-butanediol/poly(ethylene glycol-co-propylene
glycol/polycaprolactone]; poly(ethylene-co-methacrylic acid);
poly(isobutylene-co-maleic acid) sodium salt;
poly(ethylene-co-methacrylic acid) zinc salt;
poly(4-styrenesulfonic acid-co-maleic acid) sodium salt;
poly(acrylonitrile-co-butadiene-co-acrylic acid), glycidyl
methacrylate diester; poly(urea-co-formaldehyde), butylated
solution; poly(ethylene-co-methyl acrylate-co-glycidyl
methacrylate); poly[(phenyl glycidyl ether)-co-dicyclopentadiene];
poly[(o-cresyl glycidyl ether)-co-formaldehyde];
poly(urea-co-formaldehyde), methylated; poly(acrylic acid-co-maleic
acid) solution; poly(3-hydroxybutyric acid-co-3-hydroxyvaleric
acid); poly(p-toluenesulfonamide-co-formaldehyde);
poly(styrene-co-allyl alcohol);
poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-styrene);
poly(acrylonitrile-co-butadiene); poly(4-vinylphenol-co-methyl
methacrylate);
poly[dimethylsiloxane-co-methyl(3-hydroxypropyl)siloxane]-graft-poly(ethy-
lene-ran-propylene glycol) methyl ether; poly(hexafluoropropylene
oxide-co-difluoromethylene oxide) monoamidosilane;
poly(dimethylamine-co-epichlorohydrin-co-ethylenediamine) solution;
poly(ethylene-co-butyl acrylate-co-maleic anhydride);
poly(trimellitic anhydride chloride-co-4,4'-methylenedianiline);
poly[methylmethacrylate-co-(Disperse Orange 3 acrylamide)];
poly[((S)-(
)-1-(4-Nitrophenyl)-2-pyrrolidinemethyl)methacrylate-co-methylmethacrylat-
e];
poly[(propylmethacryl-heptaisobutyl-PSS)-co-(t-butylmethacrylate)];
poly[(methylmethacrylate)-co-(2-naphthylmethacrylate)];
poly[methylmethacrylate-co-(fluoresceinO-acrylate)];
poly[methylmethacrylate-co-(fluoresceinO-methacrylate)];
poly{[2-[2',5'-bis(2''-ethylhexyloxy)phenyl]-1,4-phenylenevinylene]-co-[2-
-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene]};
poly[(methylmethacrylate)-co-(Disperse Red 1 acrylate)];
poly(4-hydroxy benzoic acid-co-ethylene terephthalate);
poly(vinylidene chloride-co-acrylonitrile);
poly(dimethylsiloxane-co-diphenylsiloxane), dihydroxy terminated;
poly(1,4-butylene adipate-co-1,4-butylene succinate), extended with
1,6-diisocyanatohexane; poly(dicyclopentadiene-co-p-cresol);
poly[ethyl acrylate-co-methacrylic
acid-co-3-(1-isocyanato-1-methylethyl)-.alpha.-methylstyrene],
adduct with ethoxylated nonylphenol solution;
poly(epichlorohydrin-co-ethylene oxide); poly(Bisphenol
A-co-4-nitrophthalic anhydride-co-1,3-phenylenediamine);
poly(ethylene-co-methyl acrylate-co-acrylic acid);
poly(propylene-co-1-butene); Nylon 6/66; poly(ethylene-co-acrylic
acid) sodium salt; poly(ethylene-co-vinyl acetate-co-carbon
monoxide); poly(melamine-co-formaldehyde), methylated/butylated
(55/45); poly(maleic acid-co-olefin) sodium salt solution;
poly(tetrafluoroethylene oxide-co-difluoromethylene oxide)
.alpha.,.omega.-diisocyanate; poly(lauryl methacrylate-co-ethylene
glycol dimethacrylate); poly[(phenyl isocyanate)-co-formaldehyde];
poly[2,6-bis(hydroxymethyl)-4-methylphenol-co-4-hydroxybenzoic
acid]; poly(tetrafluoroethylene oxide-co-difluoromethylene oxide)
.alpha.,.omega.-dicarboxylic acid;
poly[methylmethacrylate-co-(Disperse yellow 7 acrylate)];
poly[(methylmethacrylate)-co-(9H-carbazole-9-ethylacrylate)];
poly[methylmethacrylate-co-(N-(1-naphthyl)-N-phenylmethacrylamide)];
poly[(methylmethacrylate)-co-(Disperse Red 1 methacrylate)];
poly(L-lactide-co-caprolactone-co-glycolide);
poly[methylmethacrylate-co-(7-(4-trifluoromethyl)coumarin
acrylamide)];
poly[dimethylsiloxane-co-methyl(3,3,3-trifluoropropyl)siloxane];
poly[dimethylsiloxane-co-methyl(stearoyloxyalkyl)siloxane];
poly(hexafluoropropylene oxide-co-difluoromethylene oxide) alcohol,
ethoxylated phosphate; poly(ethylene-co-1,2-butylene) mono-ol;
poly[dimethylsiloxane-co-methyl(3-hydroxypropyl)siloxane]-graft-tetrakis(-
1,2-butylene glycol); poly(1,4-butylene
adipate-co-polycaprolactam); poly(vinyl acetate-co-crotonic acid);
poly(tert-butyl acrylate-co-ethyl acrylate-co-methacrylic acid);
poly(1-vinylpyrrolidone-co-styrene); poly(tetrafluoroethylene
oxide-co-difluoromethylene oxide)-.alpha.,.omega.-bis(methyl
carboxylate); poly(vinylidene chloride-co-methyl acrylate);
poly(acrylonitrile-co-vinylidene chloride-co-methyl methacrylate);
poly(styrene-co-maleic anhydride), partial cyclohexyl/isopropyl
ester, cumene terminated; poly(4-ethylstyrene-co-divinylbenzene);
poly(dimethylsiloxane-co-dimer acid), bis(perfluorododecyl)
terminated; poly(styrene-co-maleic anhydride), partial propyl
ester, cumene terminated; poly(dimer acid-co-ethylene glycol),
hydrogenated; poly(ethylene-co-glycidyl methacrylate);
poly[dimethylsiloxane-co-methyl(3-hydroxypropyl)siloxane]-graft-poly(ethy-
lene glycol) 3-aminopropyl ether; poly(dimer
acid-co-1,6-hexanediol-co-adipic acid), hydrogenated;
poly(3,3',4,4'-biphenyltetracarboxylic
dianhydride-co-1,4-phenylenediamine), and amic acid solution; and
poly[N,N'-bis(2,2,6,6-tetramethyl-4-piperidinyl)-1,6-hexanediamine-co-2,4-
-dichloro-6-morpholino-1,3,5-triazine].
In particular, the block copolymer can include an ABC triblock
structure having a poly-butylacrylate segment, a poly-ethylacrylate
segment, and a poly-methacrylic acid segment, for example. The
block copolymer can include a diblock and/or triblock copolymer
having two or more different poly-aliphatic-acrylate segments. In
addition, the block copolymer can include a diblock and/or triblock
copolymer having two or more poly-alkyl-acrylate segments.
In addition, the block copolymer can be used with, or in some
embodiments replaced with, a detergent and/or a lipid. For example,
the detergents can include, but are not limited to, AOT, brij
family, Igepal family, triton family, SDS, and derivatives of each.
In particular, the detergents can include, dioctyl sulfosuccinate
sodium salt, polyethylene glycol dodecyl ether, octylphenoxy
polyethoxyethanol, octylphenyl-polyethylene glycol,
t-octylphenoxypolyethoxyethanol, polyethylene glycol
tert-octylphenyl ether,
4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, dodecyl
sulfate sodium salt, and glycolic acid ethoxylate octyl ether.
Further, the block copolymer can include lipids such as, but not
limited to, lipid-PEG, natural lipids, synthetic lipids,
sphingolipids, and derivatives of each.
The nanostructure can be attached to a probe molecule. The probe
molecule can be any molecule capable of being linked to the
nanostructure either directly or indirectly via a linker. The probe
molecule can be attached by any stable physical or chemical
association to the nanostructure directly or indirectly by any
suitable means.
In one embodiment, the probe molecule has an affinity for one or
more target molecules (e.g., cancer cell) for which detection
(e.g., determining the presence of and/or proximal position within
the vessel (body)) is desired. If, for example, the target molecule
is a nucleic acid sequence, the probe molecule should be chosen so
as to be substantially complementary to the target molecule
sequence, such that the hybridization of the target and the probe
occurs. The term "substantially complementary," means that the
probe molecules are sufficiently complementary to the target
sequences to hybridize under the selected reaction conditions.
The probe molecule and the target molecule can include, but are not
limited to, polypeptides (e.g., protein such as, but not limited to
an antibody (monoclonal or polyclonal)), nucleic acids (both
monomeric and oligomeric), polysaccharides, sugars, fatty acids,
steroids, purines, pyrimidines, drugs (e.g., small compound drugs),
ligands, or combinations thereof.
Use of the phrase "polypeptide" or "protein" is intended to
encompass a protein, a glycoprotein, a polypeptide, a peptide, and
the like, whether isolated from nature, of viral, bacterial, plant,
or animal (e.g., mammalian, such as human) origin, or synthetic,
and fragments thereof. A preferred protein or fragment thereof
includes, but is not limited to, an antigen, an epitope of an
antigen, an antibody, or an antigenically reactive fragment of an
antibody.
Use of the phrase "nucleic acid" is intended to encompass DNA and
RNA, whether isolated from nature, of viral, bacterial, plant or
animal (e.g., mammalian, such as a human) origin, synthetic,
single-stranded, double-stranded, comprising naturally or
non-naturally occurring nucleotides, or chemically modified.
In addition, the probe can also include, but is not limited to, a
drug, a therapeutic agent, radiological agent, a small molecule
drug, and combinations thereof, that can be used to treat the
target molecule and/or the associated disease and condition of
interest. The drug, therapeutic agent, and radiological agent can
be selected based on the intended treatment as well as the
condition and/or disease to be treated. In an embodiment, the
nanostructure can include two or more probes used to treat a
condition and/or disease.
The following is a nonlimiting illustrative list of probes that can
be used: PROLEUKIN.TM., CAMPATH.TM., PANRETIN.TM., ZYLOPRIM.TM.,
HEXALEN.TM., ETHYOL.TM., ARIMIDEX.TM., TRISENOX.TM., ELSPAR.TM.,
TICE BCG.TM., TARGRETIN.TM., BLENOXANE.TM., BUSULFEX.TM.,
MYLERAN.TM., METHOSARB.TM., XELODA.TM., PARAPLATIN.TM., BCNU.TM.,
BICNU.TM., GLIADEL WAFER.TM., CELEBREX.TM., LEUKERAN.TM.,
PLATINOL.TM., LEUSTATIN,-2-CDA.TM., CYTOXAN, NEOSAR.TM., CYTOXAN
INJECTION.TM., CYTOXAN TABLET.TM., CYTOSAR-U.TM., DEPOCYT.TM.,
DTIC-DOME.TM., COSMEGEN.TM., ARANESP.TM., DANUOXOME.TM.,
DAUNORUBICIN.TM., CERUBIDINE.TM., ONTAK.TM., ZINECARD.TM.,
TAXOTERE.TM., ADRIAMYCIN, RUBEX.TM., ADRIAMYCIN PFS
INJECTIONINTRAVENOUS INJECTION.TM., DOXIL.TM., DROMOSTANOLONE.TM.,
MASTERONE.TM., ELLIOTT'S B SOLUTION.TM., ELLENCE.TM., EPOGEN.TM.,
EMCYT.TM., ETOPOPHOS.TM., VEPESID.TM., AROMASIN.TM., NEUPOGEN.TM.,
FUDR.TM., FLUDARA.TM., ADRUCIL.TM., FASLODEX.TM., GEMZAR.TM.,
MYLOTARG.TM., ZOLADEX IMPLANT.TM., ZOLADREX.TM., HYDREA.TM.,
ZEVALIN.TM., IDAMYCIN.TM., IFEX.TM., GLEEVEC.TM., ROFERON-A.TM.,
INTRON A.TM., CAMPTOSAR.TM., FEMARA.TM., WELLCOVORIN,
LEUCOVORIN.TM., LEUCOVORIN.TM., ERGAMISOL.TM., CEEBU.TM.,
MUSTARGEN.TM., MEGACE.TM., ALKERAN.TM., PURINETHOL.TM., MESNEX.TM.,
METHOTREXATE.TM., UVADEX.TM., MUTAMYCIN.TM., MITOZYTREX.TM.,
LYSODREN.TM., NOVATRONE.TM., DURABOLIN-50.TM., VERLUMA.TM.,
NEUMEGA.TM., ELOXATIN.TM., PAXENE.TM., TAXOL.TM., AREDIA.TM.,
ADAGEN (PEGADEMASE BOVINE).TM., ONCASPAR.TM., NEULASTA.TM.,
NIPENT.TM., VERCYTE.TM., MITHRACIN.TM., PHOTOFRIN.TM.,
MATULANE.TM., ATABRINE.TM., ELITEK.TM., RITUXAN.TM., PROKINE.TM.,
ZANOSAR.TM., SCLEROSOL.TM., NOLVADEX.TM., TEMODAR.TM., VUMON.TM.,
TESLAC.TM., THIOGUANINE.TM., THIOPLEX.TM., HYCAMTIN.TM.,
FARESTON.TM., BEXXAR.TM., HERCEPTIN.TM., VESANOID.TM., URACIL
MUSTARD CAPSULES.TM., VALSTAR.TM., VELBAN.TM., ONCOVIN.TM.,
NAVELBINE.TM., and ZOMETAT.TM..
In an embodiment, the nanostructure can include at least two
different types of probes, one being a targeting probe that targets
certain cells or compounds associated with a condition and/or
disease, while the second probe is a drug used to treat the
disease. In this manner, the nanostructure acts as a detection
component, a delivery component to the cells of interest, and a
delivery component for the treatment agent. The detection of the
nanospecies can be used to ensure the delivery of the nanostructure
to its intended destination as well as the quantity of
nanostructures delivered to the destination.
The present disclosure provides methods of fabricating the
nanostructures. See, Current Opinion in Biotechnology 2002, 13,
40-46; Nature Biotechnology 2004, 22, 969-976 both of which are
incorporated herein by reference. An exemplary method is described
in Example 1 below.
The present disclosure provides methods of detecting one or more
target molecules in a sample or a subject (e.g., mammal, human,
cat, dog, horse, etc.), and in particular, detect the target
molecule in vivo. For example, the nanostructure can be used to
detect the presence of a tumor in an animal using the
nanostructures, as described in more detail in Example 1.
It should be noted that the nanospecies and block copolymers can
self assemble into two dimensional or three dimensional
microstructures via interactions such as, but not limited to,
hydrophobic interactions, hydrophilic interactions, charge-charge
interactions, .pi.-stacking interactions, and combinations thereof.
The self-assembly can be performed in a solution or emulsion, or on
a substrate. The microstructure can be an ordered structure or a
random structure. The microstructure can be composed of at least
one nanoparticle and one block copolymer, or composed of multiple
nanospecies and multiple block copolymers.
It should also be noted that preformed microstructures could be
doped with one or more types of nanostructures. In particular,
preformed microstructures prepared with block-copolymers (e.g.,
porous microstructures of one of many shapes (e.g., spherical)) can
be doped with nanostructures via interactions such as, but not
limited to, hydrophobic interactions, hydrophilic interactions,
charge-charge interactions, and combinations thereof, depending on
the nanostructures surface coating and block copolymer chemical
composition.
As mentioned above, it should also be noted that nanostructures
could be used for the detection of, as part of treatment (e.g.,
drug delivery), as an indication of delivery to one or more targets
(e.g., cancers), and combinations thereof, conditions and/or
diseases such as, but not limited to, cancers, tumors, neoplastic
diseases, autoimmune diseases, inflammatory diseases, metabolic
conditions, neurological and neurodegenerative diseases, viral
diseases, dermatological diseases, cardiovascular diseases, an
infectious disease, and combinations thereof.
In one embodiment, a single nanospecies coated with block
copolymers, or nanoparticle-polymer composites containing one or
more nanospecies, can be injected into subjects (e.g., humans,
domesticated animals, and cattle) as a probe or contrast reagent
for detection of primary tumors. These nanostructures can be linked
to a bio-compatible compounds (e.g., PEG and dextran) for
long-circulating "passive targeting" reagents, and/or linked to
bio-affinity probes (e.g., antibody, antigen, peptide,
oligonucleotide, small molecule ligand, and drugs) for "active"
targeting of primary tumor.
It should be noted that a cell can be pre-labeled (e.g., in vitro
and in vivo) with nanostructures and/or microstructures. For
example, cells can be labeled with nanospecies-block copolymer
microstructures in vitro through immuno staining, adsorption,
microinjection, cell uptake, and the like. The cells then can be
monitored in vitro, or traced in vivo with the nanoparticles as a
tracer, fluorescence, magnetic, combinations thereof, and the
like.
It should also be noted that nanostructures and/or microstructures
can be used as an in vivo contrast reagent in the blood pool, the
liver, the spleen, the heart, the lung, and the like. For example,
nanoparticle-block copolymer microstructures can be injected into
animals and by varying their structural properties, such as size
and/or surface coating, these microstructures can preferentially
localize into a particular organ or stay in the blood stream as a
contrast reagent.
It should also be noted that block copolymers could be used to
control the degradation of nanospecies. For example, block
copolymers can be used to either protect (make bio-compatible) the
nanospecies against degradation in biological conditions,
especially for in vivo applications, or control the degradation
rate/degree of the nanostructure, by varying the molecular
structure of the block copolymer.
Cancer, as used herein, shall be given its ordinary meaning, is a
general term for diseases in which abnormal cells divide without
control. Cancer cells can invade nearby tissues and can spread
through the bloodstream and lymphatic system to other parts of the
body.
There are several main types of cancer, for example, carcinoma is
cancer that begins in the skin or in tissues that line or cover
internal organs. Sarcoma is cancer that begins in bone, cartilage,
fat, muscle, blood vessels, or other connective or supportive
tissue. Leukemia is cancer that starts in blood-forming tissue such
as the bone marrow, and causes large numbers of abnormal blood
cells to be produced and enter the bloodstream. Lymphoma is cancer
that begins in the cells of the immune system.
When normal cells lose their ability to behave as a specified,
controlled and coordinated unit, a tumor is formed. Generally, a
solid tumor is an abnormal mass of tissue that usually does not
contain cysts or liquid areas (some brain tumors do have cysts and
central necrotic areas filled with liquid). A single tumor may even
have different populations of cells within it with differing
processes that have gone awry. Solid tumors may be benign (not
cancerous), or malignant (cancerous). Different types of solid
tumors are named for the type of cells that form them. Examples of
solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias
(cancers of the blood) generally do not form solid tumors.
Representative cancers include, but are not limited to, bladder
cancer, breast cancer, colorectal cancer, endometrial cancer, head
& neck cancer, leukemia, lung cancer, lymphoma, melanoma,
non-small-cell lung cancer, ovarian cancer, prostate cancer,
testicular cancer, uterine cancer, cervical cancer, thyroid cancer,
gastric cancer, brain stem glioma, cerebellar astrocytoma, cerebral
astrocytoma, glioblastoma, ependymoma, Ewing's sarcoma family of
tumors, germ cell tumor, extracranial cancer, Hodgkin's disease,
leukemia, acute lymphoblastic leukemia, acute myeloid leukemia,
liver cancer, medulloblastoma, neuroblastoma, brain tumors
generally, non-Hodgkin's lymphoma, osteosarcoma, malignant fibrous
histiocytoma of bone, retinoblastoma, rhabdomyosarcoma, soft tissue
sarcomas generally, supratentorial primitive neuroectodermal and
pineal tumors, visual pathway and hypothalamic glioma, Wilms'
tumor, acute lymphocytic leukemia, adult acute myeloid leukemia,
adult non-Hodgkin's lymphoma, chronic lymphocytic leukemia, chronic
myeloid leukemia, esophageal cancer, hairy cell leukemia, kidney
cancer, multiple myeloma, oral cancer, pancreatic cancer, primary
central nervous system lymphoma, skin cancer, small-cell lung
cancer, among others.
A tumor can be classified as malignant or benign. In both cases,
there is an abnormal aggregation and proliferation of cells. In the
case of a malignant tumor, these cells behave more aggressively,
acquiring properties of increased invasiveness. Ultimately, the
tumor cells may even gain the ability to break away from the
microscopic environment in which they originated, spread to another
area of the body (with a very different environment, not normally
conducive to their growth) and continue their rapid growth and
division in this new location. This is called metastasis. Once
malignant cells have metastasized, achieving cure is more
difficult.
Benign tumors have less of a tendency to invade and are less likely
to metastasize. Brain tumors spread extensively within the brain
but do not usually metastasize outside the brain. Gliomas are very
invasive inside the brain, even crossing hemispheres. They do
divide in an uncontrolled manner, though. Depending on their
location, they can be just as life threatening as malignant
lesions. An example of this would be a benign tumor in the brain,
which can grow and occupy space within the skull, leading to
increased pressure on the brain.
Cardiovascular disease, as used herein, shall be given its ordinary
meaning, and includes, but is not limited to, high blood pressure,
diabetes, coronary artery disease, valvular heart disease,
congenital heart disease, arrthymia, cardiomyopathy, CHF,
atherosclerosis, inflamed or unstable plaque associated conditions,
restinosis, infarction, thromboses, post-operative coagulative
disorders, and stroke.
Inflammatory disease, as used herein, shall be given its ordinary
meaning, and can include, but is not limited to, autoimmune
diseases such as arthritis, rheumatoid arthritis, multiple
sclerosis, systemic lupus erythematosus, other diseases such as
asthma, psoriasis, inflammatory bowel syndrome, neurological
degenerative diseases such as Alzheimer's disease, Parkinson's
disease, Huntington's disease, vascular dementia, and other
pathological conditions such as epilepsy, migraines, stroke and
trauma.
Autoimmune disease, as used herein, shall be given its ordinary
meaning, and includes, but is not limited to, alopecia areata,
ankylosing spondylitis, antiphospholipid syndrome, autoimmune
Addison's disease, aplastic anemia, autoimmune hemolytic anemia,
autoimmune hepatitis, Behcet's disease, biliary cirrhosis, bullous
pemphigoid, canavan disease, cardiomyopathy, celiac
sprue-dermatitis, chronic fatigue immune dysfunction syndrome
(CFIDS), chronic inflammatory demyelinating polyneuropathy,
Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome,
cold agglutinin disease, Crohn's disease, dermatomyositis, diffuse
cerebral sclerosis of Schilder, discoid lupus, essential mixed
cryoglobulinemia, fibromyalgia-fibromyositis, Fuch's heterochromic
iridocyclitis, Graves' disease, Guillain-Barr, Hashimoto's
thyroiditis, idiopathic pulmonary fibrosis, idiopathic
thrombocytopenia purpura (ITP), IgA nephropathy, insulin dependent
diabetes, intermediate uveitis, juvenile arthritis, lichen planus,
lupus, Mnire's disease, mixed connective tissue disease, multiple
sclerosis, myasthenia gravis, nephrotic syndrome, pemphigus
vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis,
polyglandular syndromes, polymyalgia rheumatica, polymyositis and
dermatomyositis, primary Agammag-lobulinemia, primary biliary
cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome,
rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma,
Sjogren's syndrome, stiff-man syndrome, Takayasu arteritis,
temporal arteritis/giant cell arteritis, ulcerative colitis,
vasculitis, vitiligo, VKH (Vogt-Koyanagi-Harada) disease, Wegener's
granulomatosis, anti-phospholipid antibody syndrome (lupus
anticoagulant), Churg-Strauss (allergic granulomatosis),
dermatomyositis/polymyositis, Goodpasture's syndrome, interstitial
granulomatous dermatitis with arthritis, lupus erythematosus (SLE,
DLE, SCLE), mixed connective tissue disease, relapsing
polychondritis, HLA-B27 associated conditions including ankylosing
spondylitis, psoriasis, ulcerative colitis, Reiter's syndrome, and
Uveal diseases.
Viral disease, as used herein, shall be given its ordinary meaning,
and includes target viruses such as, but not limited to, paramyxo-,
picoma-, rhino-, coxsackie-, influenza-, herpes-, adeno-,
parainfluenza-, respiratory syncytial-, echo-, corona-,
Epstein-Barr-, cytomegalo-, varicella zoster, and hepatitis (e.g.,
variants including hepatitis C Virus (HCV), Hepatitis A Virus
(HAV), Hepatitis B Virus (HBV), Hepatitis D Virus (HDV), Hepatitis
E Virus (HEV), Hepatitis F Virus (HFV), Hepatitis G Virus (HGV),
Human immunodeficiency).
Neurological conditions, as used herein, shall be given its
ordinary meaning, can be generally classified into three classes:
those disease with ischemic or hypoxic mechanisms;
neurodegenerative diseases (see Adams et al, Principles of
Neurology, 1997, 6.sup.th Ed., New York, pp 1048); and neurological
and psychiatric diseases associated with neural cell death.
Diseases with ischemic or hypoxic mechanisms can be further
subclassified into general diseases and cerebral ischemia. Examples
of such general diseases involving ischemic or hypoxic mechanisms
include myocardial infarction, cardiac insufficiency, cardiac
failure, congestive heart failure, myocarditis, pericarditis,
perimyocarditis, coronary heart disease (stenosis of coronary
arteries), angina pectoris, congenital heart disease, shock,
ischemia of extremities, stenosis of renal arteries, diabetic
retinopathy, thrombosis associated with malaria, artificial heart
valves, anemias, hypersplenic syndrome, emphysema, lung fibrosis,
and pulmonary edema. Examples of cerebral ischemia disease include
stroke (as well as hemorrhagic stroke), cerebral microangiopathy
(small vessel disease), intrapartal cerebral ischemia, cerebral
ischemia during/after cardiac arrest or resuscitation, cerebral
ischemia due to intraoperative problems, cerebral ischemia during
carotid surgery, chronic cerebral ischemia due to stenosis of
blood-supplying arteries to the brain, sinus thrombosis or
thrombosis of cerebral veins, cerebral vessel malformations, and
diabetic retinopathy.
Neurodegenerative disease can include, but is not limited to,
amyotrophic lateral sclerosis (ALS), Parkinson's disease,
Huntington's disease, Wilson's disease, multi-system atrophy,
Alzheimer's disease, Pick's disease, Lewy-body disease,
Hallervorden-Spatz disease, torsion dystonia, hereditary
sensorimotor neuropathies (HMSN), Gerstmann-Straussler-Schanke-r
disease, Creutzfeld-Jakob-disease, Machado-Joseph disease,
Friedreich ataxia, Non-Friedreich ataxias, Gilles de la Tourette
syndrome, familial tremors, olivopontocerebellar degenerations,
paraneoplastic cerebral syndromes, hereditary spastic paraplegias,
hereditary optic neuropathy (Leber), retinitis pigmentosa,
Stargardt disease, and Kearns-Sayre syndrome.
Examples of neurological and psychiatric diseases associated with
neural cell death include septic shock, intracerebral bleeding,
subarachnoidal hemorrhage, multiinfarct dementia, inflammatory
diseases (e.g., vasculitis, multiple sclerosis, and
Guillain-Barre-syndrome), neurotrauma (e.g., spinal cord trauma,
and brain trauma), peripheral neuropathies, polyneuropathies,
epilepsies, schizophrenia, metabolic encephalopathies, and
infections of the central nervous system (e.g., viral, bacterial,
fungal).
EXAMPLE 1
Now having described the embodiments of the nanostructure in
general, example 1 describes some embodiments of the nanostructure
and uses thereof. The following is a non-limiting illustrative
example of an embodiment of the present invention that is described
in more detail in Gao et al, Nature Biotechnology, 22, 8 (2004),
which is incorporated herein by reference. This example is not
intended to limit the scope of any embodiment of the present
disclosure, but rather is intended to provide specific experimental
conditions and results. Therefore, one skilled in the art would
understand that many experimental conditions can be modified, but
it is intended that these modifications be within the scope of the
embodiments of the present disclosure.
Multifunctional nanoparticle probes based on semiconductor quantum
dots (QDs) have been developed for cancer targeting and imaging in
living animals. The structural design involves encapsulating
luminescent QDs with an ABC triblock copolymer, and linking this
amphiphilic polymer to tumor-targeting ligands and drug-delivery
functionalities. In vivo targeting studies of human prostate cancer
growing in nude mice indicate that the QD probes can be delivered
to tumor sites by both enhanced permeation and retention and by
antibody binding to cancer-specific cell surface biomarkers. The
use of both subcutaneous injection of QD-tagged cancer cells and
systemic injection of multifunctional QD probes resulted in the
sensitive and multicolor fluorescence imaging of cancer cells under
in vivo conditions. This example also reports the integration of a
whole-body macro-illumination system with wavelength-resolved
spectral imaging for efficient background removal and precise
delineation of weak spectral signatures. These results raise new
possibilities for ultrasensitive and multiplexed imaging of
molecular targets in vivo.
Results
Probe Design: Bioconjugated QD probes for in vivo cancer targeting
and imaging were designed by using drug delivery and targeting
principles. As schematically illustrated in (FIG. 3A), core-shell
CdSe--ZnS quantum dots are protected by both a coordinating ligand
(TOPO) and an amphiphilic polymer coating. Due to strong
hydrophobic interactions between TOPO and the polymer hydrocarbon,
these two layers "bond" to each other and form a hydrophobic
protection structure that is resistant against hydrolysis and
enzymatic degradation even under complex in vivo conditions. In
contrast to simple polymers and amphiphilic lipids used in previous
studies, the methods described herein use a high-molecular-weight
(MW=about 100 kD) copolymer with an elaborate ABC triblock
structure and a grafted 8-carbon (C-8) alkyl side chain (FIG. 3B).
This triblock polymer includes a polybutylacrylate segment
(hydrophobic), a polyethylacrylate segment (hydrophobic), a
polymethacrylic acid segment (hydrophilic), and a hydrophobic
hydrocarbon side chain. A key finding is that this polymer can
disperse and encapsulate single TOPO-capped QDs via a spontaneous
self-assembly process. As a result, the QDs are protected to such a
degree that their optical properties (e.g., absorption spectra,
emission spectra, and fluorescence quantum yields) did not change
in a broad range of pH (1 to 14) and salt conditions (0.01 to 1 M)
or after harsh treatment with 1.0 M hydrochloric acid (PEG-linked
QDs).
Dynamic light scattering (DLS) measurement indicates that the
assembled QD probes have a hydrodynamic radius of about 10 through
15 nm (depending on attached ligands). This value agrees with a
compact probe structure consisting of a 5-nm QD core (2.5 nm
radius), a 1-nm TOPO cap, a 2-nm thick polymer layer, and a 4-5-nm
PEG/antibody layer. It has been suggested that the hydrodynamic
radii of QDs could be considerably larger than their TEM "dry"
radii, but the reported TEM values do not represent the true
physical sizes of organic-coated QDs. The reason is that organic
materials (such as TOPO, polymers, and conjugated biomolecules) are
not electron-dense enough for TEM visualization on the nanometer
scale. Since QDs are tightly protected from contacting the outside
environment, their hydrodynamic behavior is mainly controlled by
the surface-coating layer. As such, the polymer-coated quantum dots
should behave similarly as standard polymer micelles or
nanoparticles, and there is no fundamental reason for coated QDs to
have unusual hydrodynamic properties in comparison with
macromolecules and nanoparticles.
Based on the geometric/size constraints and the ligand coupling
efficiencies (about 40-50%, experimentally determined by using
fluorescently labeled ligands), it has been estimated that each dot
contains about 200 TOPO molecules, about 4 to 5 triblock copolymer
molecules, about 5 to 6 PEG molecules, and about 5 to 6 antibody
molecules. High-sensitivity fluorescence imaging showed "blinking"
signals when a dilute solution (10.sup.-12 M) of the QD
bioconjugate was spread on a glass surface. This blinking behavior
is characteristic of single quantum systems such as single dye
molecules and single QDs, indicating that the triblock copolymer
has efficiently dispersed the dots into single particles.
Preliminary TEM results also revealed that the QD probes consisted
of single particles, with little or no aggregation. It is worth
noting, however, that QD blinking has no adverse implications for
in vivo tumor imaging because the tumor cells are labeled with a
large population (up to millions) of QDs, far from the single-dot
regime.
At the current level of PEG conjugation, it does not interfere with
antibody binding, as confirmed by positive cellular staining. At
higher PEG densities or longer chains, significant interference
with ligand binding could occur, as reported previously for
pegylated liposomes. To reduce interference, the targeting ligands
could be attached to the distal termini of PEG. The fully exposed
ligands, however, could elicit nonspecific cellular uptake or an
immune response, thus reducing the probe's biocompatibility and
duration of circulation in vivo.
Tumor Targeting: Under in vivo conditions, QD probes can be
delivered to tumors by both a passive targeting mechanism and an
active targeting mechanism (FIG. 3C). In the passive mode,
macromolecules and nanometer-sized particles are accumulated
preferentially at tumor sites through an enhanced permeability and
retention (EPR) effect. This effect is believed to arise from two
factors: (a) angiogenic tumors that produce vascular endothelial
growth factors (VEGF) that hyperpermeabilize the tumor-associated
neovasculatures and cause the leakage of circulating macromolecules
and small particles; and (b) tumors lack an effective lymphatic
drainage system, which leads to subsequent macromolecule or
nanoparticle accumulation. For active tumor targeting,
antibody-conjugated quantum dots have been used to target a
prostate-specific cell surface antigen, PSMA. Previous research has
identified PSMA as a cell surface marker for both prostate
epithelial cells and neovascular endothelial cells. PSMA has been
selected as an attractive target for both imaging and therapeutic
intervention of prostate cancer. Accumulation and retention of PSMA
antibody at the site of tumor growth is the basis of
radioimmunoscintigraphic scanning (e.g., ProstaScint scan) and
targeted therapy for human prostate cancer metastasis.
The QD probes conjugated to a PSMA monoclonal antibody, J591, which
recognizes the extracellular domain of PSMA, were first evaluated
for binding to PSMA in prostate cancer cell lines.
Immunocytochemical data confirmed strong and specific binding of
the PSMA Ab J591-conjugated QD probes to a human prostate cancer
cell line, C4-2, which is known to express PSMA on the cell surface
(FIG. 4, top panels). Control studies using QD-PEG (without
antibody) showed only a low level of nonspecific cell binding to
C4-2 cells (FIG. 4, middle panels). Additional control studies
using PC-3 cells, a PSMA negative human prostate cancer cell line,
also showed the absence of QD binding (FIG. 4, lower panels). These
results establish that the PSMA antibody-QD conjugates retain their
PSMA binding activity and specificity.
To investigate the behavior of QD-PSMA Ab conjugated probes in
living animals, the following were examined in the present study:
their specific uptake and retention, background or nonspecific
uptake, blood clearance, and organ distribution as well as their
relationship to QD surface modifications. FIGS. 5A and 5B show
comparative histological data of a tumor xenograft (FIG. 5B) and
six normal host organs (FIG. 5A) obtained from a nude mouse after a
single tail vein administration of QD-PSMA Ab conjugate. As seen
from the characteristic red-orange fluorescence of quantum dots,
nonspecific QD uptake and retention took place primarily in the
liver and the spleen, with little or no QD accumulation in the
brain, the heart, the kidney, or the lung. This pattern of in vivo
organ uptake and distribution is similar to that of dextran-coated
magnetic iron oxide nanoparticles. For polymer-encapsulated QDs
with excess COOH groups, no tumor targeting was observed,
indicating nonspecific organ uptake and rapid blood clearance. For
polymer-encapsulated QDs with surface PEG groups, the rate of organ
uptake was reduced and the length of blood circulation was
improved, leading to slow accumulation of the nanoparticles in the
tumors. For QDs encapsulated by PEG and bioconjugated with PSMA
antibody, the nanoparticles were delivered and retained by the
tumor xenografts, but nonspecific liver and spleen uptake was still
apparent.
Passive tumor targeting was observed only with an increased dose of
QD-PEG conjugate (6 nmol injected plus a 24-hour latent period of
probe circulation). In contrast, this same dose of QD-COOH
conjugate was found to have little accumulation in tumors due to
passive targeting following the same length of circulation in
athymic hosts. This low efficiency of QD uptake and retention is
likely due to the excess negative charges on the probe surface
(free carboxylic acid groups on the polymer coating), which is
known to reduce the rate of probe extravasation and its subsequent
accumulation into tumor xenografts.
In vivo Cancer Imaging: FIGS. 6A through 6D depict spectral imaging
results obtained from PSMA-Ab QD probes injected into the tail vein
of a tumor-bearing mouse and a control mouse (no tumor). The
original image (FIG. 6A) shows QD signals at one tumor site among
an autofluorescence background (mouse skin). Using spectral
unmixing algorithms, the fluorescence background signals (FIG. 6B)
can be separated from the QD signals. (FIG. 6C). The composite
image (FIG. 6D) clearly shows the whole animal and the tumor site.
The enhanced contrast in the bottom right image indicates that the
QD probes can be visualized against an essentially black
background, with little or no interference from the mouse
autofluorescence. Results from separate tests using quantum dots
excited in vitro indicate that spectral imaging techniques can be
used to unmix multiple fluorescent signals that differ by as little
as 5 nm in peak position (results not shown). Thus, the ability to
exclude interference from autofluorescence and the capability of
resolving multiple simultaneous labels suggest that spectral
imaging will have considerable utility when combined with
quantum-dot-based labeling strategies.
The present study has further examined how functional groups on the
QD probe surface affect in vivo imaging results. FIG. 7 compares
the in vivo imaging results from three types of surface
modifications: COOH groups, PEG groups, and PEG plus PSMA Ab. In
agreement with histological examinations, no tumor signals were
detected with the COOH probe; only weak tumor signals were observed
with the PEG probe (passive targeting); and intense signals were
detected in the PEG-PSMA Ab conjugated probe (active targeting).
This comparison provides further evidence that active tumor
targeting by using a tumor-specific ligand is much faster and more
efficient than passive targeting based on tumor permeation, uptake
and retention.
Probe Brightness and Spectral Comparison with GFP: Since
genetically encoded fluorescent proteins such as GFP have been used
to tag cells for in vivo cancer imaging, it is important to compare
the detection sensitivity and spectral features of GFP and QD
probes. For this purpose, QDs were first linked to a translocation
peptide (such as HIV Tat or polyarginine), and were delivered into
living cancer cells. Similar peptides have been used to deliver
magnetic nanoparticles into living cells for in vivo monitoring of
cell migration and integration. Fluorescence intensity measurement
indicates that as many as three million QDs can be delivered into
each cancer cell. Surprisingly, this level of QD loading did not
affect cell viability and growth, because the implantation of
QD-tagged cancer cells led to usual tumor growth in animal
models.
FIG. 8A shows in vivo imaging data for the same number (about 1000)
of QD-tagged cells and GFP stably transfected cells that were
injected into each side of a host mouse. Although the QD-tagged
cells and the GFP transfected cells were similarly bright in cell
cultures (two images on the right), only the QD signal was observed
in vivo (orange glow on the right flank). No GFP signals could be
discerned at the injection site (circle on the left flank). This
result does not provide an absolute intensity comparison between
GFP and QDs because several factors (such as optical density and
tissue scattering) are difficult to normalize or calibrate.
Instead, it is a qualitative spectral comparison demonstrating that
the emission spectra of QDs can be shifted away from the
autofluorescence, allowing spectroscopic detection at low signal
intensities. In contrast, organic dyes and fluorescent proteins
give rise to small Stokes shifts, resulting GFP emission and
background fluorescence in the same spectral region. The brightness
and spectral shifting advantages of QDs are further shown in FIGS.
9A and 9B and 10A and 10B.
Another important feature is the large absorption coefficients of
QDs, which makes them brighter probes under photon-limited in vivo
conditions (where light intensities are severely attenuated by
scattering and absorption). To appreciate this feature, the
photophysics of quantum dots and organic dyes can be compared. In
theory, the lifetime-limited emission rates for single quantum dots
are 5-10 times lower than those of single organic dyes because of
their longer excited state lifetimes (20-50 ns). In practice,
however, fluorescence imaging usually operates under
absorption-limited conditions, in which the rate of absorption is
the main limiting factor of fluorescence emission. Since the molar
extinction coefficients (0.5-2.times.10.sup.6 M.sup.-1 cm.sup.-1)
of QDs are about 10-50 times larger than that (5-10.times.10.sup.4
M.sup.-1 cm.sup.-1) of organic dyes, the QD absorption rates will
be 10-50 times faster than that of organic dyes at the same
excitation photon flux. Due to this increased rate of light
emission, single QDs appear 10-20 times brighter than organic dyes,
a result that has been experimentally confirmed by the current
literature.
The present study has further explored multicolor in vivo imaging
with QD-encoded microbeads. For this purpose, three samples of 0.5
.mu.m polymer beads, each doped with green, yellow or red QDs, were
injected into a mouse model at three different locations, similar
to previous reports of using fluorescent beads for cell
differentiation and trafficking studies. Due to the usually large
Stokes shifts and broad excitation profiles of QDs, all three
colors were observed simultaneously in the same mouse and with a
single light source (FIG. 8B).
Discussion
Prior to this work, several groups have reported the use of QDs for
sensitive bioassays and cellular imaging, but a significant loss of
fluorescence has been noted upon the administration of quantum dots
into live animals. While the exact origin of this signal loss is
still not clear, recent research suggests that the surface ligands
and coatings are slowly degraded in body fluids, leading to surface
defects and fluorescence quenching. This mechanism is supported by
the observation that the surface defects can be annealed by
continuous laser excitation, and the loss of QD fluorescence can be
partially restored (involving surface structural changes). The QD
probes reported in this work represent a significant improvement
because they are highly stable against in vivo degradation. An
important feature is a high-molecular-weight triblock copolymer,
which completely encapsulates TOPO-QDs and forms a stable
hydrophobic protection layer around single QDs.
On the hydrophilic surface of this polymer layer, there is a large
number of functional groups (e.g., about 400 to 500 carboxylic
acids groups), which allows the attachment of both diagnostic and
therapeutic agents. With small-molecule ligands such as synthetic
organic molecules, short oligonucleotides and peptides, many copies
of the same ligand can be linked to single dots, leading to
multivalent QD-target binding. Previous research has shown that
properly designed multivalent ligands can increase the binding
affinity by 10 orders of magnitude. Using colloidal gold
nanoparticles linked to oligos at high surface densities, it has
been demonstrated that the sequence selectivity of DNA
hybridization can be improved by 100 to 1000 times (sharper melting
curves). Research has also shown that QD-peptide conjugates exhibit
exquisite binding specificity, most likely due to multivalent
peptide binding to protein targets distributed on the surface of
tumor vasculature. This novel feature is not available with organic
dyes and fluorescent proteins, and could allow the design of
multivalent QD probes to target cancer cells based on the density
and distribution of biomarkers on the cell surface. This might
offer a new strategy for cancer molecular diagnosis and therapy
because truly unique cancer biomarkers are often not available or
are present at extremely low concentrations.
In addition, the polymer-encapsulated QD probes are in an excellent
size range for in vivo tumor targeting. With small peptide-dye
conjugates, rapid extravasation often leads to blood clearance of
the probe in less than one minute. The circulation or retention
time can be improved by attaching small probes to macromolecules or
nanoparticles, a strategy widely used in drug delivery research.
Indeed, the described work indicates that PEG-shielded QDs are able
to circulate in blood for as long as about 48-72 hours, with a half
decay time of about 5-8 hours. At the same time, these probes are
small enough for efficient binding to cell surface receptors, for
internalization through endocytosis or peptide translocation, and
for passing through the nuclear pores to enter the cell nucleus
(using nuclear-localization peptides) (FIG. 8A, top right).
However, the penetration depth of QDs into solid tumors will be
limited, at least in part, by their nanometer sizes.
The unique optical properties of QDs also provide new opportunities
for multicolor imaging and multiplexing. For example, multicolor
imaging will allow intensity ratioing, spatial colocalization, and
quantitative target measurements at metastatic tumor sites. Optical
encoding strategies are also possible based on the use of multiple
colors and multiple intensity levels. This combinatorial approach
has been demonstrated for tagging a large number of genes,
proteins, and small-molecule libraries. In addition to wavelength
and intensity, lifetime fluorescence imaging represents a new
dimension. Because the excited state lifetimes (about 20-50 ns) of
QDs are nearly one order magnitude longer than that of organic dyes
(about 2-5 ns), QD probes should be suitable for fluorescence
lifetime imaging (FLIM) of cells, tissue specimens, and living
animals.
The current use of orange/red-emitting quantum dots is not
optimized for tissue penetration or imaging sensitivity. Extensive
work in tissue optics has shown deep tissue imaging (millimeters to
centimeters) requires the use of far-red and near-infrared light in
the spectral range of 650-900 nm. This wavelength range provides a
"clear" window for in vivo optical imaging because it is separated
from the major absorption peaks of blood and water. Based on tissue
optical calculations, it is estimated that the use of
near-infrared-emitting quantum dots should improve the tumor
imaging sensitivity by at least 10-fold, allowing sensitive
detection of 10-100 cancer cells. Toward this goal, recent research
has prepared a new class of alloyed semiconductor quantum dots
consisting of cadmium selenium telluride, with tunable fluorescence
emission up to 850 nm and quantum yields up to 60%. Together with
core-shell CdTeCdSe type-II materials, the use of
near-infrared-emitting QDs should bring major improvements in
tissue penetration depth and cell detection sensitivity.
A remaining issue is the QD's toxicity and metabolism in vivo.
Recent work indicates that CdSe QDs are highly toxic to cells under
UV illumination for extended periods of time. This is
understandable because UV-irradiation often dissolves the
semiconductor particles, releasing toxic cadmium ions into the
medium. In the absence of UV irradiation, the present work shows
that QDs with a stable polymer coating are essentially nontoxic to
cells (no effect on cell division or ATP production). Current
literature shows that in vivo studies also confirmed the nontoxic
nature of stably protected QDs. This is perhaps not surprising
because the polymer protection layer is so stable that the QD core
would not be exposed to the outside environment. Consistent with
this conclusion, previous research has shown that the uptake of
dextran-protected iron oxide nanoparticles (up to 10 million
particles per cell) does not significantly reduce cell viability,
and that the injection of micelle-protected QDs (up to 2 billion
per embryo cell) does not affect frog embryo development. In this
work, up to 3 million QDs in a single cancer cell did not
appreciably reduce its viability or growth.
At the present, however, little is known about the mechanism of
metabolism or clearance of QD probes injected into living animals.
For the polymer-encapsulated QDs, chemical or enzymatic
degradations of the semiconductor cores are unlikely to occur. But
the polymer-protected QDs might be cleared from the body by slow
filtration and excretion through the kidney.
In conclusion, the present study involves the development of a new
class of polymer-encapsulated and bioconjugated QD probes for
cancer targeting and imaging in vivo. These probes are bright,
stable, and have a versatile triblock copolymer structure that is
well suited for conjugation to additional diagnostic and
therapeutic agents. In vivo imaging results indicate the QD probes
can be targeted to tumor sites through both passive and active
mechanisms, but passive targeting is much slower and less efficient
than active targeting. When combined with wavelength-resolved
imaging, the QD probes allow sensitive and multicolor imaging of
cancer cells in living animals. The use of near-infrared-emitting
quantum dots should improve both the tissue penetration depth and
imaging sensitivity. In accordance with the described study,
quantum dots could be integrated with targeting, imaging, and
therapeutic agents to develop "smart" nanostructures for
noninvasive imaging, diagnosis, and treatment of cancer,
cardiovascular plaques, and neurodegenerative disease.
Methods: Animal use protocols were reviewed and approved by the
Institutional Animal Care and Use Committee of Emory
University.
Materials: Except noted otherwise, all chemicals and biochemicals
were purchased from Sigma-Aldrich (St. Louis, Mo.) and were used
without further purification. A monoclonal antibody (J591) to
prostate-specific membrane antigen (PSMA) was a kind gift from
Millennium Pharmaceuticals (Cambridge, Mass.). Membrane
translocation peptides (Tat and polyarginine, with a c-terminal
biotin for conjugation to strepavidin-QD) was synthesized and
purified by Invitrogen (Carlsbad, Calif.). Core-shell quantum dots
(ZnS-capped CdSe) were synthesized according to literature
procedures. A high-temperature coordinating solvent,
tri-n-octylphosphine oxide (TOPO), was used for the synthesis,
leading to high-quality QDs that were capped by a monolayer of TOPO
molecules. These dots were highly fluorescent (about 60% quantum
yields) and monodispersed (about 5% size variations). QD-encoded
microbeads were prepared by using 0.5 .mu.m mesoporous microbeads
in butanol, and were isolated and purified as reported
previously.
A triblock copolymer consisting of a poly-butylacrylate segment, a
poly-ethylacrylate segment, and a poly-methacrylic acid segment was
purchased from Sigma (St. Louis, Mo.). At a molecular weight of
about 100,000 daltons, this polymer contains more than 1000 total
monomer units, with a weight distribution of 23% methacrylic acid
and 77% combined butyl and ethyl acrylates. For encapsulating QDs,
about 25% of the free carboxylic acid groups were derivatized with
octylamine (a hydrophobic side chain). Thus, the original polymer
dissolved in dimethylformamide (DMF) was reacted with n-octylamine
at a polymer/octylamine molar ratio of 1:40, using ethyl-3-dimethyl
amino propyl carbodiimide (EDAC, 3-fold excess of n-octylamine) as
a cross-linking reagent. The product yields were generally greater
than 90% due to the high EDAC coupling efficiency in DMF
(determined by a change of the free octylamine band in thin layer
chromatography). The reaction mixture was dried with a ratovap
(Rotavapor R-3000, Buchi Analytical Inc, Delaware). The resulting
oily liquid was precipitated with water, and was rinsed with water
5 times to remove excess EDAC and other by-products. After vacuum
drying, the octylamine-grafted polymer was re-suspended in an
ethanol/chloroform mixture, and was stored for use.
Surface modification and bioconjugation: Using a 3:1 (v/v)
chloroform/ethanol solvent mixture, TOPO-capped quantum dots were
encapsulated by the amphiphilic tri-block polymer. A polymer-to-QD
ratios of 5 to 10 was used because molecular geometry calculations
indicated that at least 4 polymer molecules would be required to
completely encapsulate one quantum dot. Indeed, stable
encapsulation (e.g., no aggregation) was not achieved at
polymer/dot ratios less than 4:1. After vacuum drying, the
encapsulated dots were suspended in a polar solvent (aqueous buffer
or ethanol), and were purified by gel filtration. Standard
procedures were then used to crosslink free carboxylic acid groups
(about 100 on each polymer molecule) with amine-containing ligands
such as amino-PEGs (Sunbio, Korea), peptides, and antibodies.
Briefly, the polymer-coated dots were activated with 1 mM EDAC at
pH 6 for 30 min. After purification, the activated dots were
reacted with amino-PEG at a QD/PEG molar ratio of 1:50 at pH 8 for
2 hours, generating PEG-linked probes. Alternatively, the activated
dots were reacted with PEG at a reduced QD/PEG ratio of 1:6 at pH 8
for 20 min, and then with a tumor-targeting antibody at a
QD/antibody molar ratio of 1:15 for 2 hours. The final QD
bioconjugates were purified by column filtration or
ultracentrifugation at 100,000 g for 30 min. After resuspension in
PBS buffer (pH 7), aggregated particles were removed by
centrifugation at 6000 g for 10 min.
QD-streptavidin was prepared by using the same cross-linking
reagent (1-mM EDAC) and under the same experimental conditions
(1:15 QD/strepavidin molar ratio, pH 8, room temperature, and 2
hours) as for QD-antibody conjugates. After purification by column
filtration, QD-streptavidin was mixed with biotinylated Tat (or
polyarginine) at a QD/peptide molar ratio of 1:20, and was
incubated at room temperature in PBS buffer (pH 7) for 30 min with
occasional sonication. The product was purified by filtration
column chromatography. Conjugation of Tat or polyarginine to QDs
was confirmed by using dual-labeled peptides (biotin at one end and
an organic dye separate from QD fluorescence at the other end). The
peptide-QD conjugate was added to cell culture media to a final
concentration of 20 nM, and was incubated at 37.degree. C. from 1
hour to 24 hours.
Fluorescence imaging: In vivo fluorescence imaging was accomplished
by using a macro-illumination system (Lightools Research,
Encinitas, Calif.), designed specifically for small animal studies.
True-color fluorescence images were obtained using dielectric
long-pass filters (Chroma Tech, Brottleboro, Vt.) and a digital
color camera (Optronics, Magnafire SP, Olympus America, Melville,
N.Y.). Wavelength-resolved spectral imaging was carried out by
using a spectral imaging system (CRI, Inc., Woburn, Mass.)
comprising a optical head that includes a liquid crystal tunable
filter (LCTF, with a bandwidth of 20 nm and a scanning wavelength
range of 400 to 720 nm), an optical coupler and a cooled,
scientific-grade monochrome CCD camera, along with image
acquisition and analysis software. The tunable filter was
automatically stepped in 10 nm increments from 580 to 700 nm while
the camera captured images at each wavelength with constant
exposure. Overall acquisition time was about 10 seconds. The 13
resulting TIFF images were loaded into a single data structure in
memory, forming a spectral stack with a spectrum at every pixel.
With spectral imaging software, small but meaningful spectral
differences could be rapidly detected and analyzed.
Autofluorescence spectra and quantum dot spectra were manually
selected from the spectral image using the computer mouse to select
appropriate regions. Spectral unmixing algorithms (available from
CRI, Inc., Woburn, Mass.) were applied to create the unmixed images
of "pure" autofluorescence and "pure" quantum dot signal, a
procedure that takes about one second on a typical personal
computer. When appropriately generated, the autofluorescence image
should be uniform in intensity regardless of the presence or
absence of quantum-dot signals (as is the case in FIG. 6A through
6D). The identification of valid spectra for unmixing purposes need
only be performed initially, as the spectra can be saved in
spectral libraries and re-used on additional spectral stacks.
Cells and tissue sections were examined by using an inverted
Olympus microscope (IX-70) equipped with a digital color camera
(Nikon D1), a broad-band ultraviolet (330-385 nm) light source
(100-W mercury lamp), and a long-pass interference filter (DM 400,
Chroma Tech, Brattleboro, Vt.). Wavelength-resolved spectra were
obtained by using a single-stage spectrometer (SpectraPro 150,
Roper Scientific, Trenton, N.J.).
Cell, tissue, and whole-animal studies: Both human breast cancer
cells (MDA-MB-231) and PSMA-positive human prostate cancer cells
(C4-2) were used for implantation into immuno-compromised Balb/c
nude mice. These two cell lines were maintained in RPMI and T
media, respectively with 10% fetal bovine serum. Conventional
immunohistochemical procedures were used to determine the binding
of PSMA Ab-QD conjugate to C4-2 prostate cancer cells, utilizing
both PEG-QD (no antibody) and PC-3 cells (no PSMA antigen) as
negative controls. For pre-tagging of cancer cells, QDs were linked
to a transduction peptide such as HIV Tat or polyarginine as noted
above, and were delivered into living cancer cells by incubation at
37.degree. C. After one hour incubation, each cell was found to
contain more than one million QDs, and with overnight incubation,
essentially all the QDs were localized in the cell nucleus.
Using protocols approved by the Institutional Animal Care and Use
Committee of Emory University, about one million tumor cells were
injected into 6-8 week old nude mice subcutaneously (Charles River,
Wilmington, Mass.). Tumor growth was monitored daily until it
reached the acceptable sizes. The mice were divided into 2 groups
for passive and active targeting studies. QD bioconjugates were
injected into the tail vein, at 0.4 nmole for active targeting or
6.0 nmol (about 15 times more) for passive targeting. The mice were
placed under anesthesia by injection of a Ketamine and Xylazine
mixture intraperitioneally at a dosage of 95 mg/kg and 5 mg/kg,
respectively. In a dark box, illumination was provided by
fiberoptic lighting, and a long pass filter was used to reject
scattered excitation light and to pass Stokes-shifted QD
fluorescence. Fluorescent images were recorded by scientific-grade
CCDs. After whole-body imaging, the mice were sacrificed by
CO.sub.2 overdose. Tumor and major organs were removed and frozen
for histological QD uptake and distribution studies. Tissue
collections were cryosectioned into 5-10 .mu.m thickness sections,
fixed with acetone at 0.degree. C., and examined with an
epi-fluorescence microscope (Olympus Axiovert, Melville, N.Y.).
It should be emphasized that the above-described embodiments of the
present disclosure are merely possible examples of implementations,
and are merely set forth for a clear understanding of the
principles of this disclosure. Many variations and modifications
may be made to the above-described embodiment(s) of the disclosure
without departing substantially from the spirit and principles of
the disclosure. All such modifications and variations are intended
to be included herein within the scope of this disclosure and
protected by the following claims.
* * * * *
References